JP7522106B2 - Pharmaceutical compositions comprising bispecific anti-CD37 antibodies - Google Patents

Description

FIELD OF THEINVENTION The present invention relates to pharmaceutical compositions comprising bispecific antibodies that specifically bind to the human CD37 antigen. The present invention particularly relates to pharmaceutical compositions comprising CD37-specific bispecific antibody molecules that bind to different epitopes of the human CD37 antigen, which bispecific antibody molecules have enhanced Fc-Fc interactions upon binding to CD37 on the cell surface and thus enhanced effector functions. The present invention also relates to the use of pharmaceutical compositions containing these molecules for the treatment of cancer and other diseases.

2. Background of the Invention Leukocyte antigen CD37 "CD37" (also known as GP52-40, tetraspanin-26, or TSPAN26) is a transmembrane protein of the tetraspanin superfamily (Maecker et al., FASEB J. 1997;11:428-442 (Non-Patent Document 1)). In normal physiology, CD37 is reported to be expressed on B cells during the pre-B to peripheral mature B cell stage, but is absent on plasma cells (Link et al., J Pathol. 1987;152:12-21 (Non-Patent Document 2)). The CD37 antigen is only weakly expressed on T cells and myeloid cells, such as monocytes, macrophages, dendritic cells, and granulocytes (Schwartz-Albiez et al., J. Immunol 1988;140(3):905-914 (Non-Patent Document 3)). CD37 is widely expressed on malignant cells in a variety of B-cell leukemias and lymphomas, including non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL) (Moore et al. J Immunol. 1986;137(9):3013 (Non-Patent Document 4)).

Several antibody-based CD37 targeting agents are being evaluated as potential therapeutics for B cell and other malignancies. These include, for example, radioimmunoconjugates such as Betalutin®, antibody-drug conjugates such as IMGN529 and AGS-67E, and reshaped or Fc-modified antibodies such as otlertuzumab and BI 836826 (Robak and Robak, Expert Opin Biol Ther 2014;14(5):651-61). Anti-CD37 antibodies have been proposed for use as therapeutic agents in these and other formats (see, e.g., WO2012/135740 (Patent Document 1), WO2012/007576 (Patent Document 2), WO2011/112978 (Patent Document 3), WO2009/126944 (Patent Document 4), WO2011/112978 (Patent Document 5) and EP2241577 (Patent Document 6)).

Betalutin is the murine anti-CD37 antibody rilotomab (previously HH1/tetulomab) conjugated to 177-lutetium. Betalutin is rapidly internalized, inhibits B cell growth in vitro, and extends survival in the intravenous Daudi-SCID model (Dahle et al 2013, Anticancer Res 33:85-96).

IMGN529 is an ADC consisting of the K7153A antibody conjugated to the maytansinoid DM1 via an SMCC linker. The K7153 antibody has been reported to induce apoptosis in CD37 expressing Ramos cells in the absence of cross-linking. It also induced CDC and ADCC in Burkitt's lymphoma cell lines, but its ability to induce CDC was much less than that of rituximab (Deckert et al, Blood 2013; 122(20):3500-10). These Fc-mediated effector functions of K7153A are retained in the DM-1 conjugated antibody.

Agensys is developing AGS-67E, a human anti-CD37 IgG2 mAb conjugated to monomethyl auristatin E. AGS67E induces potent cytotoxicity and apoptosis (Pereira et al, Mol Cancer Ther 2015; 14(7): 1650-1660 (Non-Patent Document 8)).

Otlertuzumab (originally known as TRU-016) is a SMIP (small modular immunopharmaceutical product). SMIPs are disulfide-linked single-chain protein dimers consisting of one antigen-binding VH/VL, a connecting hinge region, and an Fc (fragment crystallizable) region (CH2-CH3). Its mechanism of action is the induction of apoptosis and ADCC, but not CDC (Zhao et al 2007, Blood 110(7), 2569-2577).

mAb37.1/BI 836826 is a chimeric antibody engineered for high affinity binding to FcγRIIIa (CD16a) (Heider et al 2011, Blood 118: 4159-4168). It has proapoptotic activity independent of IgG Fc cross-linking, but the proapoptotic activity is enhanced by cross-linking. It shows potent ADCC in CD37 ⁺ B cell lines and primary CLL cells.

Despite these and other advances in the art, however, there remains a need for improved anti-CD37 antibodies and stable pharmaceutical formulations thereof for the treatment of cancer and other diseases.

WO2012/135740 WO2012/007576 WO2011/112978 WO2009/126944 WO2011/112978 EP2241577

Maecker et al., FASEB J. 1997;11:428-442 Link et al., J Pathol. 1987;152:12-21 Schwartz-Albiez et al., J. Immunol 1988;140(3):905-914 Moore et al. J Immunol. 1986;137(9):3013 Robak and Robak, Expert Opin Biol Ther 2014;14(5):651-61 Dahle et al 2013, Anticancer Res 33:85-96 Deckert et al, Blood 2013; 122(20):3500-10 Pereira et al, Mol Cancer Ther 2015; 14(7): 1650-1660 Zhao et al 2007, Blood 110(7), 2569-2577 Heider et al 2011, Blood 118: 4159-4168

PCT/EP2018/058479 (unpublished), which is incorporated herein by reference, provides anti-CD37 antibodies for the treatment of cancer and/or other diseases, including bispecific antibodies having binding arms derived from two parent antibodies that bind to different epitopes on CD37, the bispecific antibodies having enhanced CDC and/or ADCC compared to a combination of two parent monoclonal antibodies that bind to the different epitopes and/or either parent monoclonal antibody alone. PCT/EP2018/058479 further provides bispecific antibodies that bind to two different epitopes on CD37, the bispecific antibodies having enhanced Fc-Fc interactions upon binding to CD37 on the cell membrane compared to a bispecific antibody of the same isotype and having the same binding arms as the bispecific antibody.

The present invention provides a stable pharmaceutical composition comprising a bispecific antibody having binding arms that bind to different epitopes on CD37.

Thus, in one aspect, the present invention provides a method for producing a composition comprising:
a) bispecific antibodies;
b) histidine buffer;
c) 50-300 mM sugar and/or 50-300 mM polyol, and
d) 0.01 to 0.1% polysorbate 80
A pharmaceutical composition comprising:
the pH of the composition is between 4.5 and 6.8; and the bispecific antibody comprises a first and a second antigen-binding region that binds to human CD37 having the sequence of SEQ ID NO: 62, and a first and a second Fc region of a human immunoglobulin, the first and the second Fc region comprising one or more amino acid mutations that enhance the Fc-Fc interaction between the bispecific antibodies upon binding to a membrane-bound target compared to the Fc-Fc interaction between the bispecific antibodies not comprising the mutations;
The first antigen binding region comprises the CDR sequence:
VH CDR1 sequence as set forth in SEQ ID NO: 16;
VH CDR2 sequence as set forth in SEQ ID NO: 17;
VH CDR3 sequence as set forth in SEQ ID NO: 18;
The VL CDR1 sequence according to SEQ ID NO: 20;
a VL CDR2 sequence that is a KAS, and
The second antigen-binding region comprises a VL CDR3 sequence as set forth in SEQ ID NO: 21, and the second antigen-binding region comprises the CDR sequence:
VH CDR1 sequence as set forth in SEQ ID NO: 23;
VH CDR2 sequence as set forth in SEQ ID NO: 24;
VH CDR3 sequence as set forth in SEQ ID NO: 25;
The VL CDR1 sequence set forth in SEQ ID NO: 27;
a VL CDR2 sequence which is YAS, and
The pharmaceutical composition comprises the VL CDR3 sequence set forth in SEQ ID NO: 31.

In one aspect of the invention, the pharmaceutical composition comprises:
e) bispecific antibodies;
f) histidine buffer;
g) 50-300 mM sugar and/or 50-300 mM polyol, and
h) 0.01 to 0.1% polysorbate 80
Including,
The pH of the composition is between 4.5 and 6.8; and the bispecific antibody comprises a first antigen-binding region comprising a heavy chain set forth in SEQ ID NO:124 and a light chain set forth in SEQ ID NO:119, and a second antigen-binding region comprising a heavy chain set forth in SEQ ID NO:125 and a light chain set forth in SEQ ID NO:126.

The present invention also provides a stable pharmaceutical composition comprising an antibody having a binding arm that binds to CD37.

Thus, in one aspect, the present invention provides a method for producing a composition comprising:
a) an antibody;
b) histidine buffer;
c) 50-300 mM sugar and/or 50-300 mM polyol, and
d) 0.01 to 0.1% polysorbate 80
A pharmaceutical composition comprising:
the antibody comprises a first antigen-binding region that binds human CD37 having the sequence of SEQ ID NO: 62, and a first and a second Fc region of a human immunoglobulin, the first and second Fc regions comprising one or more amino acid mutations that enhance Fc-Fc interactions between the antibodies upon binding to a membrane-bound target compared to Fc-Fc interactions between bispecific antibodies not having the mutations, the first antigen-binding region comprising the CDR sequence:
VH CDR1 sequence as set forth in SEQ ID NO: 16;
VH CDR2 sequence as set forth in SEQ ID NO: 17;
VH CDR3 sequence as set forth in SEQ ID NO: 18;
The VL CDR1 sequence according to SEQ ID NO: 20;
a VL CDR2 sequence that is a KAS, and
The pharmaceutical composition comprises the VL CDR3 sequence set forth in SEQ ID NO: 21.

In a further aspect, the present invention provides a method for producing a method for treating a cancer cell comprising the steps of:
e) antibodies,
f) histidine buffer;
g) 50-300 mM sugar and/or 50-300 mM polyol, and
h) 0.01 to 0.1% polysorbate 80
A pharmaceutical composition comprising:
the antibody comprises a second antigen-binding region that binds human CD37 having the sequence of SEQ ID NO: 62, and a first and a second Fc region of a human immunoglobulin, the first and second Fc regions comprising one or more amino acid mutations that enhance Fc-Fc interactions between the antibodies upon binding to a membrane-bound target compared to Fc-Fc interactions between bispecific antibodies not having the mutations, the second antigen-binding region comprising the CDR sequence:
VH CDR1 sequence as set forth in SEQ ID NO: 23;
VH CDR2 sequence as set forth in SEQ ID NO: 24;
VH CDR3 sequence as set forth in SEQ ID NO: 25;
The VL CDR1 sequence set forth in SEQ ID NO: 27;
a VL CDR2 sequence which is YAS, and
The pharmaceutical composition comprises the VL CDR3 sequence set forth in SEQ ID NO: 31.

In other aspects, the present invention relates to the use of a pharmaceutical composition of the present invention for the manufacture of a medicament, and to methods of treatment comprising administering a pharmaceutical composition of the present invention.
[The present invention 1001]
a) bispecific antibodies;
b) histidine buffer;
c) 50-300 mM sugar and/or 50-300 mM polyol, and
d) 0.01 to 0.1% polysorbate 80
A pharmaceutical composition comprising:
The composition has a pH of 4.5 to 6.8; and
The bispecific antibody comprises first and second antigen-binding regions that bind human CD37 having the sequence of SEQ ID NO: 62, and first and second Fc regions of a human immunoglobulin, the first and second Fc regions comprising one or more amino acid mutations that enhance Fc-Fc interaction between the bispecific antibodies upon binding to a membrane-bound target compared to Fc-Fc interaction between bispecific antibodies not having the mutations, and the first antigen-binding region comprises the CDR sequence:
VH CDR1 sequence as set forth in SEQ ID NO: 16;
VH CDR2 sequence as set forth in SEQ ID NO: 17;
VH CDR3 sequence as set forth in SEQ ID NO: 18;
The VL CDR1 sequence according to SEQ ID NO: 20;
a VL CDR2 sequence that is a KAS, and
VL CDR3 sequence as set forth in SEQ ID NO: 21
and the second antigen binding region comprises the CDR sequence:
VH CDR1 sequence as set forth in SEQ ID NO: 23;
VH CDR2 sequence as set forth in SEQ ID NO: 24;
VH CDR3 sequence as set forth in SEQ ID NO: 25;
The VL CDR1 sequence set forth in SEQ ID NO: 27;
a VL CDR2 sequence which is YAS, and
VL CDR3 sequence as set forth in SEQ ID NO: 31
The pharmaceutical composition comprising:
[The present invention 1002]
a) an antibody;
b) histidine buffer;
c) 50-300 mM sugar and/or 50-300 mM polyol, and
d) 0.01 to 0.1% polysorbate 80
A pharmaceutical composition comprising:
The composition has a pH of 4.5 to 6.8; and
The antibody comprises a first antigen-binding region that binds human CD37 having the sequence of SEQ ID NO: 62, and a first and a second Fc region of a human immunoglobulin, the first and second Fc regions comprising one or more amino acid mutations that enhance Fc-Fc interactions between the antibodies upon binding to a membrane-bound target compared to Fc-Fc interactions between bispecific antibodies not having the mutations, and the first antigen-binding region comprises the CDR sequence:
VH CDR1 sequence as set forth in SEQ ID NO: 16;
VH CDR2 sequence as set forth in SEQ ID NO: 17;
VH CDR3 sequence as set forth in SEQ ID NO: 18;
The VL CDR1 sequence according to SEQ ID NO: 20;
a VL CDR2 sequence that is a KAS; and
VL CDR3 sequence according to SEQ ID NO: 21
The pharmaceutical composition comprising:
[The present invention 1003]
a) an antibody;
b) histidine buffer;
c) 50-300 mM sugar and/or 50-300 mM polyol, and
d) 0.01 to 0.1% polysorbate 80
A pharmaceutical composition comprising:
The composition has a pH of 4.5 to 6.8; and
The antibody comprises a second antigen-binding region that binds human CD37 having the sequence of SEQ ID NO: 62, and a first and a second Fc region of a human immunoglobulin, the first and second Fc regions comprising one or more amino acid mutations that enhance Fc-Fc interactions between the antibodies upon binding to a membrane-bound target compared to Fc-Fc interactions between bispecific antibodies not having the mutations, and the second antigen-binding region comprises the CDR sequence:
VH CDR1 sequence as set forth in SEQ ID NO: 23;
VH CDR2 sequence as set forth in SEQ ID NO: 24;
VH CDR3 sequence as set forth in SEQ ID NO: 25;
The VL CDR1 sequence set forth in SEQ ID NO: 27;
a VL CDR2 sequence which is YAS, and
VL CDR3 sequence as set forth in SEQ ID NO: 31
The pharmaceutical composition comprising:
[The present invention 1004]
The pharmaceutical composition of the present invention 1001 comprising 5-100 mg/mL of the bispecific antibody, such as 10-50 mg/mL, such as 10-30 mg/mL, such as 20 mg/mL of the bispecific antibody.
[The present invention 1005]
The pharmaceutical composition of the invention 1002 or 1003, comprising 5-100 mg/mL of the antibody, such as 10-50 mg/mL, such as 10-30 mg/mL, such as 20 mg/mL of the antibody.
[The present invention 1006]
Any of the aforementioned pharmaceutical compositions according to the invention comprising 10 to 100 mM histidine, such as 10 to 50 mM, such as 10 to 30 mM, such as 20 mM histidine.
[The present invention 1007]
Any of the preceding pharmaceutical compositions of the invention which comprise a sugar which is sucrose and preferably comprises 75-275 mM sucrose, such as 100-250 mM, such as 100 mM sucrose or 250 mM sucrose.
[The present invention 1008]
1007. A pharmaceutical composition according to the present invention, which does not contain a polyol.
[The present invention 1009]
8. The pharmaceutical composition of any of claims 1001-1007, comprising a polyol which is sorbitol or mannitol, preferably comprising 75-275 mM sorbitol or 75-275 mM mannitol, such as 100-250 mM sorbitol or 100-250 mM mannitol, for example 100 mM sorbitol or 100 mM mannitol or 250 mM sorbitol or 100 mM mannitol.
[The present invention 1010]
Any of the aforementioned pharmaceutical compositions according to the invention comprising 0.01 to 0.05% polysorbate 80, such as 0.01% to 0.04%, for example 0.02% or 0.04% polysorbate 80.
[The present invention 1011]
Any of the aforementioned pharmaceutical compositions of the invention, wherein the pH is between 5.5 and 6.5, such as 5.5 or 6.5.
[The present invention 1012]
Any of the above pharmaceutical compositions of the invention further comprising sodium chloride, such as 25 to 250 mM sodium chloride, such as 100 to 150 mM sodium chloride, such as 100 mM or 150 mM sodium chloride.
[The present invention 1013]
Any of the aforementioned pharmaceutical compositions of the invention further comprising arginine, such as 25 to 200 mM arginine, such as 50 to 100 mM arginine, such as 75 mM arginine.
[The present invention 1014]
a) 20 mg/mL bispecific antibody, 20 mM histidine, 250 mM sucrose, and 0.02% or 0.04% polysorbate 80 at pH 5.5-6.5; or
b) 20 mg/mL of bispecific antibody, 20 mM histidine, 100 mM sucrose, 0.02% or 0.04% polysorbate 80, and 100-150 mM, preferably 100 mM, sodium chloride at pH 5.5-6.5; or
c) 20 mg/mL of bispecific antibody, 20 mM histidine, 100 mM sucrose, 0.02% or 0.04% polysorbate 80, 75 mM arginine, and 100-150 mM, preferably 100 mM, sodium chloride at pH 5.5-6.5; or
d) pH 5.5-6.5, containing 20 mg/mL bispecific antibody, 20 mM histidine, 100 mM sucrose, 0.02% or 0.04% polysorbate 80, and 75 mM arginine.
Any of the pharmaceutical compositions of the present invention.
[The present invention 1015]
In aqueous solution:
a) 20 mg/mL bispecific antibody, 20 mM histidine, 250 mM sucrose, and 0.02% or 0.04% polysorbate 80 at pH 5.5-6.5; or
b) 20 mg/mL of bispecific antibody, 20 mM histidine, 100 mM sucrose, 0.02% or 0.04% polysorbate 80, and 100-150 mM, preferably 100 mM, sodium chloride at pH 5.5-6.5; or
c) 20 mg/mL of bispecific antibody, 20 mM histidine, 100 mM sucrose, 0.02% or 0.04% polysorbate 80, 75 mM arginine, and 100-150 mM, preferably 100 mM, sodium chloride at pH 5.5-6.5; or
d) 20 mg/mL bispecific antibody, 20 mM histidine, 100 mM sucrose, 0.02% or 0.04% polysorbate 80, and 75 mM arginine at pH 5.5-6.5.
Any of the pharmaceutical compositions of the present invention.
[The present invention 1016]
The first antigen-binding region has the following VH and VL sequences:
a) the VH sequence set forth in SEQ ID NO: 15 and the VL sequence set forth in SEQ ID NO: 19, or
b) a VH sequence having at least 90% identity, such as at least 95% identity, such as at least 98% identity, such as at least 99% identity, and a VL sequence having at least 90% identity, such as at least 95% identity, such as at least 98% identity, such as at least 99% identity, to the VH and VL sequences of SEQ ID NOs: 15 and 19, provided that the sequences of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of said first antigen-binding region remain as defined in invention 1001 and 1002.
Any of the pharmaceutical compositions of the present invention comprising:
[The present invention 1017]
The first antigen-binding region has the following VH and VL sequences:
a) the VH sequence set forth in SEQ ID NO: 15 and the VL sequence set forth in SEQ ID NO: 127, or
b) a VH sequence having at least 90% identity, such as at least 95% identity, such as at least 98% identity, such as at least 99% identity, and a VL sequence having at least 90% identity, such as at least 95% identity, such as at least 98% identity, such as at least 99% identity, to the VH and VL sequences of SEQ ID NOs: 15 and 127, provided that the sequences of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of said first antigen-binding region remain as defined in invention 1001 and 1002.
Any of the pharmaceutical compositions of the present invention comprising:
[The present invention 1018]
the second antigen-binding region
a) the VH sequence set forth in SEQ ID NO: 22 and the VL sequence set forth in SEQ ID NO: 29, or
b) a VH sequence having at least 90% identity, such as at least 95% identity, such as at least 98% identity, such as at least 99% identity, and a VL sequence having at least 90% identity, such as at least 95% identity, such as at least 98% identity, such as at least 99% identity, to the VH and VL sequences of SEQ ID NOs: 22 and 29, provided that the sequences of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of said second antigen-binding region remain as defined in invention 1001 and 1003.
Any of the aforementioned pharmaceutical compositions of the invention comprising a VH and VL sequence selected from the group comprising:
[The present invention 1019]
Any of the aforementioned pharmaceutical compositions of the present invention, wherein one or more of the Fc-Fc interaction enhancing mutations in the first and second Fc regions are amino acid substitutions.
[The present invention 1020]
Any of the aforementioned pharmaceutical compositions of the invention, wherein the one or more Fc-Fc interaction enhancing mutations in the first and second Fc regions are amino acid substitutions at one or more positions corresponding to amino acid positions 430, 440, and 345 in human IgG1 using the EU numbering system.
[The present invention 1021]
Any of the aforementioned pharmaceutical compositions of the present invention, comprising at least one substitution in said first and second Fc regions selected from the group including E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y, and S440W.
[The present invention 1022]
Any of the aforementioned pharmaceutical compositions of the invention comprising at least one substitution in said first and second Fc regions selected from E430G or E345K, preferably E430G.
[The present invention 1023]
Any of the pharmaceutical compositions of the present invention, wherein the Fc-Fc interaction enhancing mutations in the first and second Fc regions are identical substitutions in the first and second Fc regions.
[The present invention 1024]
Any of the aforementioned pharmaceutical compositions of the invention, wherein the bispecific antibody is of the IgG1, IgG2, IgG3, or IgG4 isotype, or a combination thereof, preferably of the IgG1 isotype.
[The present invention 1025]
Any of the aforementioned pharmaceutical compositions of the present invention, wherein the bispecific antibody is a full-length antibody.
[The present invention 1026]
Any of the aforementioned pharmaceutical compositions of the present invention, wherein the bispecific antibody is a human antibody, a humanized antibody, or a chimeric antibody, or a combination thereof.
[The present invention 1027]
a) the first Fc region comprises an additional mutation corresponding to F405L when using EU numbering in human IgG1, and the second Fc region comprises an additional mutation corresponding to K409R when using EU numbering in human IgG1, or
b) the second Fc region comprises an additional mutation corresponding to F405L when using EU numbering in human IgG1, and the first Fc region comprises an additional mutation corresponding to K409R when using EU numbering in human IgG1;
Any of the pharmaceutical compositions of the present invention.
[The present invention 1028]
Any of the aforementioned pharmaceutical compositions of the invention, wherein the bispecific antibody consists of a heavy chain set forth in SEQ ID NOs: 118 and 120 and a light chain set forth in SEQ ID NOs: 119 and 121, wherein the heavy chain set forth in SEQ ID NO: 118 forms an antigen-binding region with the light chain set forth in SEQ ID NO: 119, and the heavy chain set forth in SEQ ID NO: 120 forms an antigen-binding region with the light chain set forth in SEQ ID NO: 121.
[The present invention 1029]
Any of the aforementioned pharmaceutical compositions of the invention, wherein the bispecific antibody consists of a heavy chain set forth in SEQ ID NOs: 124 and 125 and a light chain set forth in SEQ ID NOs: 119 and 126, wherein the heavy chain set forth in SEQ ID NO: 124 forms an antigen-binding region with the light chain set forth in SEQ ID NO: 119, and the heavy chain set forth in SEQ ID NO: 125 forms an antigen-binding region with the light chain set forth in SEQ ID NO: 126.
[The present invention 1030]
Any of the preceding pharmaceutical compositions of the invention having enhanced CDC, or enhanced CDC and ADCC effector function, compared to the same bispecific molecule not having the Fc-Fc interaction enhancing mutation.
[The present invention 1031]
Any of the pharmaceutical compositions according to the invention for use as a medicament.
[The present invention 1032]
Any of the preceding pharmaceutical compositions of the invention for use in the treatment of cancer or an autoimmune disease or an inflammatory disorder.
[The present invention 1033]
Any of the preceding pharmaceutical compositions of the present invention for use in the treatment of allergies, transplant rejection, or B-cell malignancies, such as non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), mantle cell lymphoma (MCL), plasma cell leukemia (PCL), diffuse large B-cell lymphoma (DLBCL), or acute lymphoblastic leukemia (ALL).
[The present invention 1034]
A pharmaceutical composition for use according to any of claims 1031-1033 which is administered parenterally, for example subcutaneously, intramuscularly or intravenously.
[The present invention 1035]
A pharmaceutical composition for use according to any of claims 1031-1034 in combination with one or more further therapeutic agents.
[The present invention 1036]
The pharmaceutical composition for use according to any of claims 1031 to 1035, wherein said one or more further therapeutic agents are selected from the group comprising doxorubicin, cisplatin, bleomycin, carmustine, cyclophosphamide, chlorambucil, bendamustine, vincristine, fludarabine, ibrutinib, and anti-CD20 antibodies, such as rituximab or ofatumumab.
[The present invention 1037]
The additional therapeutic agent is
i) the VH CDR1 sequence set forth in SEQ ID NO: 75;
VH CDR2 sequence as set forth in SEQ ID NO: 76;
VH CDR3 sequence as set forth in SEQ ID NO: 77;
The VL CDR1 sequence set forth in SEQ ID NO: 79;
a VL CDR2 sequence which is a DAS, and
VL CDR3 sequence as set forth in SEQ ID NO: 80;
ii) the VH CDR1 sequence set forth in SEQ ID NO: 82;
VH CDR2 sequence as set forth in SEQ ID NO: 83;
VH CDR3 sequence as set forth in SEQ ID NO: 84;
VL CDR1 sequence as set forth in SEQ ID NO: 85;
a VL CDR2 sequence which is a DAS, and
VL CDR3 sequence as set forth in SEQ ID NO: 86;
iii) the VH CDR1 sequence set forth in SEQ ID NO: 95;
VH CDR2 sequence as set forth in SEQ ID NO: 96;
VH CDR3 sequence as set forth in SEQ ID NO: 97;
VL CDR1 sequence as set forth in SEQ ID NO: 99;
a VL CDR2 sequence which is an ATS, and
VL CDR3 sequence as set forth in SEQ ID NO: 100;
iv) the VH CDR1 sequence set forth in SEQ ID NO: 88;
VH CDR2 sequence as set forth in SEQ ID NO: 89;
VH CDR3 sequence as set forth in SEQ ID NO: 90;
VL CDR1 sequence as set forth in SEQ ID NO: 92;
a VL CDR2 sequence which is a DAS, and
The VL CDR3 sequence set forth in SEQ ID NO: 93; and
v) the VH CDR1 sequence set forth in SEQ ID NO: 102;
VH CDR2 sequence as set forth in SEQ ID NO: 103;
VH CDR3 sequence as set forth in SEQ ID NO: 104;
The VL CDR1 sequence set forth in SEQ ID NO: 106;
a VL CDR2 sequence that is QMS, and
VL CDR3 sequence set forth in SEQ ID NO: 107
A pharmaceutical composition for use in accordance with the present invention 1035 or 1036, which is an anti-CD20 antibody capable of binding to human CD20, comprising a CDR sequence selected from the group consisting of:
[The present invention 1038]
Use of any of the pharmaceutical compositions according to claims 1001 to 1030 for the manufacture of a medicament.
[The present invention 1039]
The use of the compound of the present invention 1038 for the manufacture of a medicament for the treatment of cancer, an autoimmune disease, or an inflammatory disease.
[The present invention 1040]
Use of 1038 of the present invention for the manufacture of a medicament for the treatment of allergies, transplant rejection, or B-cell malignancies, such as non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), mantle cell lymphoma (MCL), plasma cell leukemia (PCL), diffuse large B-cell lymphoma (DLBCL), or acute lymphoblastic leukemia (ALL).
[The present invention 1041]
The use of any of claims 1038 to 1040, wherein said pharmaceutical composition is administered parenterally, for example subcutaneously, intramuscularly or intravenously.
[The present invention 1042]
The use of any of 1038-1041 in combination with one or more further therapeutic agents.
[The present invention 1043]
The use of the present invention 1042, wherein said one or more further therapeutic agents are selected from the group comprising doxorubicin, cisplatin, bleomycin, carmustine, cyclophosphamide, chlorambucil, bendamustine, vincristine, fludarabine, ibrutinib, and anti-CD20 antibodies, such as rituximab or ofatumumab.
[The present invention 1044]
A method for inducing cell death or inhibiting the growth and/or proliferation of tumor cells expressing CD37, comprising administering to an individual in need thereof an effective amount of any of the pharmaceutical compositions of the present inventions 1001-1030.
[The present invention 1045]
A method for treating an individual having allergy, an autoimmune disease, an inflammatory disease, transplant rejection, or a B-cell malignancy, such as non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), mantle cell lymphoma (MCL), plasma cell leukemia (PCL), diffuse large B-cell lymphoma (DLBCL), or acute lymphoblastic leukemia (ALL), comprising administering to the individual an effective amount of any of the pharmaceutical compositions of the present invention 1001-1026.
[The present invention 1046]
The method of claim 1041 or 1042, wherein said pharmaceutical composition is administered parenterally, for example subcutaneously, intramuscularly or intravenously.
[The present invention 1047]
The method of any of claims 1044 to 1046, comprising administering in combination with said pharmaceutical composition one or more additional therapeutic agents.
[The present invention 1048]
The method of claim 1047, wherein said one or more further therapeutic agents are selected from the group comprising doxorubicin, cisplatin, bleomycin, carmustine, cyclophosphamide, chlorambucil, bendamustine, vincristine, fludarabine, ibrutinib, and anti-CD20 antibodies, such as rituximab or ofatumumab.

CDC in primary CLL tumor cells mediated by G28.1 variants. The ability of (A) IgG1-G28.1-K409R-delK, IgG1-G28.1-E345R, or IgG1-b12-E345R (cells: patient-derived, newly diagnosed/naïve (PB = peripheral blood derived)) and (B) IgG1-G28.1, IgG1-G28.1-E430G, or IgG1-b12 (cells: patient-derived, newly diagnosed/naïve (BM = bone marrow derived)) to induce CDC in primary CLL cells was measured in vitro. Data shown are % lysis measured by flow cytometric counting of the percentage of dead cells (corresponding to PI-positive cells). Quantification of CD37, CD46, CD55, and CD59 expression levels on CLL tumor cells. Expression levels of CD37, CD46, CD55, and CD59 on CLL cells from one patient (patient VM-PB0005, newly diagnosed/untreated) were measured by flow cytometry. Antigen load is shown as molecules/cell. mIgG1 is the mouse IgG1 kappa isotype control. Binding of humanized CD37 antibodies and their variants to Daudi cells. Binding of IgG1-004-H5L2, IgG1-004-H5L2-E430G, IgG1-005-H1L2, IgG1-005-H1L2-E430G, IgG1-010-H5L2, IgG1-010-H5L2-E430G, IgG1-016-H5L2, and IgG1-016-H5L2-E430G to Daudi cells was measured by flow cytometry. Data shown are mean fluorescence intensity (MFI) values from one representative experiment. Binding of G28.1 and 37.3 and their variants to Daudi cells. Binding of IgG1-G28.1, IgG1-G28.1-E430G, IgG1-37.3, and IgG1-37.3-E430G to Daudi cells was measured by flow cytometry. Data shown are mean fluorescence intensity (MFI) values from one representative experiment. Binding of variants of humanized CD37 antibody IgG1-016-H5L2 to Daudi cells. Binding of IgG1-016-H5L2, IgG1-016-H5L2-E430G, IgG1-016-H5L2-F405L-E430G, and IgG1-016-H5L2-LC90S-F405L-E430G to Daudi cells was measured by flow cytometry. Data shown are mean fluorescence intensity (MFI) values from one representative experiment. Binding of CD37 antibody variants to CHO cells expressing cynomolgus CD37. Binding of IgG1-004-H5L2-E430G, IgG1-005-H1L2-E430G, IgG1-010-H5L2-E430G, IgG1-016-H5L2-E430G, IgG1-G28.1, and IgG1-G28.1-E430G was measured by flow cytometry. Data shown are mean fluorescence intensity (MFI) values from one representative experiment. Figure 7: Measurement of binding competition between CD37 antibodies and CDC on Raji cells mediated by humanized CD37 antibodies, their variants, and combinations of CD37 antibodies. (A) Binding competition between IgG1-37.3-E430G, IgG1-G28.1-E430G, IgG1-004-H5L2-E430G, IgG1-005-H1L2-E430G, IgG1-010-H5L2-E430G, and IgG1-016-H5L2-E434G was measured by flow cytometry. Raji cells were incubated with unlabeled antibodies for primary binding and then incubated with Alexa Fluor 488-labeled probe antibodies. Loss of binding of A488-labeled probe antibodies after preincubation with unlabeled antibodies compared to binding of A488-labeled antibodies alone indicates binding competition between A488-labeled and unlabeled antibodies. Data shown are MESF (soluble fluorescent molecules count) duplicate values from one representative experiment. (B-G) The ability of IgG1-004-H5L2, IgG1-005-H1L2, IgG1-010-H5L2, IgG1-016-H5L2, and IgG1-37.3 (with or without the E430G mutation) and combinations thereof to induce CDC in Raji cells was measured in vitro. Data shown are % lysis measured by flow cytometric counting of the percentage of dead cells (corresponding to PI positive cells). See legend to Figure 7A. See legend to Figure 7A. See legend to Figure 7A. See legend to Figure 7A. See legend to Figure 7A. See legend to Figure 7A. Overview of binding competition between CD37 antibodies. Binding competition between IgG1-37.3-E430G, IgG1-G28.1-E430G, IgG1-004-H5L2-E430G, IgG1-005-H1L2-E430G, IgG1-010-H5L2-E430G, and IgG1-016-H5L2-E4340G to Raji cells was measured by flow cytometry using unlabeled antibodies for primary binding and an Alexa Fluor 488-labeled probe antibody to detect subsequent binding of competing antibodies. Color designation: black; simultaneous binding, white; competing for binding, grey; same antibody. CDC mediated by humanized CD37 antibodies and their variants in Daudi cells. The ability of IgG1-004-H5L2, IgG1-004-H5L2-E430G, IgG1-005-H1L2, IgG1-005-H1L2-E430G, IgG1-010-H5L2, IgG1-010-H5L2-E430G, IgG1-016-H5L2, and IgG1-016-H5L2-E430G to induce CDC in Daudi cells was measured in vitro. Data shown are % lysis measured by flow cytometric counting of the percentage of dead cells (corresponding to PI positive cells). Figure 10: CDC mediated by G28.1 and 37.3 and their variants, and by humanized CD37 antibodies with Fc-Fc interaction enhancing mutations in Daudi cells. (A) The ability of IgG1-G28.1, IgG1-G28.1-E430G, IgG1-37.3, and IgG1-37.3-E430G to induce CDC in Daudi cells was measured in vitro. Data shown are % lysis measured by flow cytometric counting of the percentage of dead cells (corresponding to PI positive cells). (B-C) The ability of (A) IgG1-010-H5L2-K409R-E430G, IgG1-010-H5L2-E345R-K409R, IgG1-010-H5L2-E345K-K409R, IgG1-010-H5L2-K409R-E430S, IgG1-010-H5L2-RRGY, and (B) IgG1-016-H5L2-LC90S-F405L-E430G, IgG1-016-H5L2-E345K-F405L, IgG1-016-H5L2-F405L-E430S, and IgG1-016-H5L2-E345R-F405L to induce CDC in Daudi cells was measured in vitro. Data shown are % lysis (maximal killing at an antibody concentration of 10 μg/mL) measured by flow cytometric counting of the percentage of dead cells (corresponding to PI positive cells) from one representative experiment. Error bars indicate within-experiment variability (performed in duplicate). See legend to Figure 10A. See legend to Figure 10A. CDC mediated by variants of the humanized antibody IgG1-016-H5L2 in Daudi cells. The ability of IgG1-016-H5L2, IgG1-016-H5L2-E430G, IgG1-016-H5L2-F405L-E430G, and IgG1-016-H5L2-LC90S-F405L-E430G to induce CDC in Daudi cells was measured in vitro. Data shown are % lysis measured by flow cytometric counting of the percentage of dead cells (corresponding to PI-positive cells). Figure 12: CDC mediated by bispecific CD37 antibodies with Fc-Fc interaction enhancing mutations, (combinations of) CD37 antibodies with Fc-Fc interaction enhancing mutations and monovalent CD37 binding antibodies with Fc-Fc interaction enhancing mutations in Daudi cells; and CDC activity of CD37 antibody variants with Fc-Fc interaction enhancing mutations and their combinations in OCI-Ly-7 cells. (A) bsIgG1-016-H5L2-LC90S-F405L-E430G x 005-H1L2-K409R-E430G, IgG1-005-H1L2-E430G, IgG1-016-H5L2-E430G, the combination of IgG1-005-H1L2-K409R-E430G and IgG1-016-H5L2-F405L-E430G, bsIgG1-b12-F405L-E430G x 005-H1L2-K409R-E430G, and bsIgG1-016-H5L2-LC90S-F405L-E430G x b12-K409R-E430G, and (B) bsIgG1 The ability of -016-H5L2-LC90S-F405L-E430G x 010-H5L2-K409R-E430G, IgG1-010-H5L2-E430G, IgG1-016-H5L2-E430G, the combination of IgG1-010-H5L2-E430G and IgG1-016-H5L2-E430G, bsIgG1-016-H5L2-LC90S-F405L-E430G x b12-K409R-E430G, and bsIgG1-b12-F405L-E430G x 010-H5L2-K409R-E430G to induce CDC in Daudi cells was measured in vitro. Data shown are % lysis measured by flow cytometry counting the percentage of dead cells (corresponding to PI positive cells). (C) CD37 bispecific antibody bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G, CD37 monospecific bivalent (monoclonal) antibodies IgG1-010-H5L2-E430G, IgG1-016-H5L2-E430G, combination of IgG1-010-H5L2-E430G and IgG1-016-H5L2-E430G, monovalent CD37 antibody bsIgG1-016-H5L2-LC90S The ability of bsIgG1-016-H5L2-LC90S-F405L-E430G×b12-K409R-E430G, bsIgG1-b12-F405L-E430G×010-H5L2-K409R-E430G, and the combination of bsIgG1-016-H5L2-LC90S-F405L-E430G×b12-K409R-E430G with bsIgG1-b12-F405L-E430G×010-H5L2-K409R-E430G to induce OCI-Ly-7 cells in vitro was measured. Data shown are % lysis measured by counting the percentage of dead cells (corresponding to PI positive cells) by flow cytometry. (D) EC50 values for CDC induction by bsIgG1-016-H5L2-LC90S-F405L-E430G × b12-K409R-E430G and bsIgG1-b12-F405L-E430G × 010-H5L2-K409R-E430G, and by IgG1-010-H5L2-E430G and IgG1-016-H5L2-E430G, determined in two independent experiments. (E) EC50 values for CDC induction by bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G, and IgG1-010-H5L2-E430G and IgG1-016-H5L2-E430G determined in three independent experiments. See legend to Figure 12A. See legend to Figure 12A. See legend to Figure 12A. See legend to Figure 12A. Figure 13: CDC mediated by bispecific CD37 antibodies and bispecific CD37 antibodies with Fc-Fc interaction enhancing mutations in Daudi cells. The ability of (A) bsIgG1-016-H5L2-F405Lx005-H1L2-K409R, and bsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G, and (B) bsIgG1-016-H5L2-F405Lx010-H5L2-K409R and bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G to induce CDC in Daudi cells was measured in vitro. Data shown are % lysis determined by counting the percentage of dead cells (corresponding to PI positive cells) by flow cytometry. See legend to Figure 13A. Figure 14: CDC mediated by bispecific CD37 antibodies with Fc-Fc interaction enhancing mutations, (combinations of) CD37 antibodies with Fc-Fc interaction enhancing mutations, and monovalent binding CD37 antibodies with Fc-Fc interaction enhancing mutations in primary CLL tumor cells. (A) bsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G, IgG1-005-H1L2-K409R-E430G, IgG1-016-H5L2-F405L-E430G, IgG1-005-H1L2-K409R-E430G and IgG1-016- (B) bsIgG1-016-H5L2-LC90S-F405L-E430G×b12-K409R-E430G, bsIgG1-b12-F405L-E430G×005-H1L2-K409R-E430G, and bsIgG1-016-H5L2-LC90S-F405L-E430G×b12-K409R-E430G, and (C) bsIgG1-016-H5L2-LC The ability of 90S-F405L-E430Gx010-H5L2-K409R-E430G, IgG1-010-H5L2-E430G, IgG1-016-H5L2-E430G, the combination of IgG1-010-H5L2-E430G and IgG1-016-H5L2-E430G, bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G, and bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G to induce CDC in primary CLL tumor cells (patient: VM-BM0091, newly diagnosed/untreated (BM = bone marrow derived)) was measured in vitro. Data shown are % lysis determined by counting the percentage of dead cells (corresponding to PI positive cells) by flow cytometry. See legend to Figure 14A. CDC mediated by bispecific CD37 antibodies with Fc-Fc interaction enhancing mutations in B cell lymphoma cell lines. The ability of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G (at a concentration of 10 μg/mL) to induce CDC in various B cell lymphoma cell lines was measured in vitro. CD37 expression levels were measured by quantitative flow cytometry and are shown as molecules/cell (mean ± SD of two experiments). Open bars indicate susceptibility to CDC mediated by bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G (>10% lysis, average of two experiments) and closed bars indicate insusceptibility to CDC mediated by bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G (<10% lysis, average of two experiments). Figure 16: ADCC mediated by bispecific CD37 antibodies with Fc-Fc interaction enhancing mutations, (combinations of) CD37 antibodies with Fc-Fc interaction enhancing mutations, and monovalent binding CD37 antibodies with Fc-Fc interaction enhancing mutations in Daudi and Raji cells. (A) bsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G, IgG1-005-H1L2-K409R-E430G, IgG1-016-H5L2-F405L-E430G, and IgG1-005-H1L2-K409R-E430G with IgG1-016-H5L2-F405L-E430G. Ability of the combinations to induce ADCC in Daudi cells, (B) bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G, IgG1-010-H5L2-E430G, IgG1-016-H5L2-E430G, and IgG1-010-H5L2-E430G with IgG1-016-H5L2-E (C) Ability of bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G, IgG1-010-H5L2-E430G, IgG1-016-H5L2-E430G, IgG1-010-H5L2-E430G, and IgG1-016 The ability of bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G and bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G to induce ADCC in Raji cells was measured in vitro using a chromium release assay. Data shown are % specific lysis; error bars indicate intra-assay variability (5 replicates (A, B) or 6 replicates (C) per data point). See legend to Figure 16A. See legend to Figure 16A. Figure 17: Quantification of CD37, CD46, CD55, and CD59 expression levels in (A) CLL, (B) FL, (C) MCL, or (D) DLBCL tumor cells. Expression levels in tumor cells were measured by flow cytometry. Antigen amounts are shown as antibody binding capacity. See legend to Figure 17A. See legend to Figure 17A. See legend to Figure 17A. Figure 18: CDC mediated by bispecific CD37 antibodies with Fc-Fc interaction enhancing mutations in primary tumor cells from CLL, FL, MCL, DLBCL or B-NHL (not further specified) patients. The ability of bsIgG1-016-H5L2-LC90S-F405Lx010-H5L2-K409R-E430G to induce CDC in tumor cells from (A) CLL, (B) FL and (C) MCL, DLBCL or B-NHL (not further specified) patients was measured by flow cytometry. CDC induction is presented as lysis (%) measured by fraction of 7-AAD-positive tumor cells using bsIgG1-016-H5L2-LC90S-F405L×010-H5L2-K409R-E430G at 100 μg/mL (A and B) or 10 μg/mL (C). See legend to Figure 18A. See legend to Figure 18A. Binding of bispecific CD37 antibodies with Fc-Fc interaction enhancing mutations to B cells in human or cynomolgus blood. Binding of Alexa-488 labeled bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G to B cells in (A) human or (B) cynomolgus blood was measured by flow cytometry. Alexa-488 labeled IgG1-b12 was used as a negative control antibody. Data are shown as geometric mean A488 fluorescence intensity values from one representative donor/animal. Error bars indicate within-experiment (duplicate measurements) variability. Cytotoxicity of bispecific CD37 antibodies with Fc-Fc interaction enhancing mutations and monoclonal CD37-specific antibodies with enhanced FcγR interaction against B cells in human or cynomolgus blood. (A) Cytotoxicity of bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G and IgG1-CD37-B2-S239D-I332E against B cells in human blood and (B) bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G against B cells in cynomolgus blood was measured in a whole blood cytotoxicity assay. IgG1-b12 was used as a negative control antibody. Data are shown as % B cell depletion from one representative donor/animal, error bars indicate within-experiment variability (duplicate measurements). Figure 21: CDC mediated by bispecific CD37 antibodies with Fc-Fc interaction enhancing mutations, CD20 specific antibodies, or their combination. (A-D) The ability of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G, ofatumumab, or their combination (at the indicated concentrations) to induce CDC in tumor cells from two CLL patients was measured ex vivo. Data are presented as the percentage of viable B cells. See legend to Figure 21A. See legend to Figure 21A. See legend to Figure 21A. Dose-effect relationship of three weekly doses of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G in the JVM-3 model. (A) Tumor growth of JVM-3 xenografts after treatment with various doses of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G or isotype control antibody (IgG1-b12). Means and SEM are shown for each group (n=10) at each time point. (B) Tumor size per mouse at day 25. Means and SEM are shown for each treatment group. Differences were analyzed by Mann-Whitney test. Statistically significant differences were indicated as follows: **: p<0.01; ***: p<0.001. Dose-effect relationship of three weekly doses of bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G in the Daudi-luc model. (A) Tumor growth (measured by luciferase activity, bioluminescence) of Daudi-luc xenografts after treatment with various doses of bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G or isotype control antibody (IgG1-b12). Means and SEM are shown for each group (n=9) at each time point. (B) Luciferase activity per mouse at day 36. Means and SEM are shown for each treatment group. Differences were analyzed by one-way ANOVA, Fisher's LSD uncorrected. Statistically significant differences were indicated as follows: **: p<0.01; ***: p<0.001. Figure 24: Plasma concentrations of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G and IgG1-b12 after intravenous injection in SCID mice. SCID mice were injected once intravenously with (A-B) 100μg (5mg/kg) or (C-D) 500μg (25mg/kg) of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G or IgG1-b12. See description of Figure 24-1. Analysis of binding of CD37 antibodies to CD37 variants with alanine mutations in the extracellular domain. The z-score (fold change) was defined as (normalized gMFI [aa position] - μ)/σ, where μ and σ are the mean and standard deviation (SD) of normalized gMFI of all variants. Residues with z-scores below -1.5 (indicated by the dotted line) were considered as "loss-of-binding mutants". Numbers on the x-axis refer to amino acid positions. Note that the x-axis is discontinuous. The left part of the axis (up to the striped line) represents aa residues in the small extracellular loop of human CD37 that are not alanine or cysteine; the right part of the axis represents aa residues in the large extracellular loop of human CD37 that are not alanine or cysteine. The dotted line indicates a z-score (fold change) of -1.5. FIG. 26: CDC mediated by a mixture of CD37 antibodies with Fc-Fc interaction enhancing mutations plus a clinically established CD20 antibody product on Raji cells. CDC-mediated Raji cell killing (% lysis expressed as PI positive cell fraction measured by flow cytometry) for antibody concentration dilution series (final concentration 10 μg/mL) of 1:0, 3:1, 1:1, 3:1, and 0:1 antibody mixtures of CD37 antibodies with Fc-Fc interaction enhancing mutations plus standard of care CD20 antibody products MabThera (rituximab), Arzerra (ofatumumab), and Gazyva (obinutuzumab, GA101): (A) with IgG1-37.3-E430G, (B) with IgG1-G28.1-E430G, (C) with IgG1-004-E430G, (D) with IgG1-005-E430G, (E) with IgG1-010-E430G, and (F) with IgG1-016-E430G. See legend to Figure 26A. See legend to Figure 26A. See legend to Figure 26A. See legend to Figure 26A. See legend to Figure 26A. Turbidity of antibody formulations. Turbidity in Nephelometric Turbidity Units (NTU) measured using a turbidimeter. Black circles represent IgG1-016-H5L2-LC90S-F405L-E430G (D1). White circles represent IgG1-010-H5L2-K409R-E430G (E1). Sub-visible particle counts in antibody formulations. Sub-visible particles in various formulations after two freeze-thaw cycles measured using a HIAC instrument. Particles larger than 2, 5, 10, or 25 micrometers were counted.

Detailed Description of the Invention
Definition
As used herein, the term "CD37" refers to the leukocyte antigen CD37, also known as GP52-40, tetraspanin-26, and TSPAN26, which is a heavily glycosylated transmembrane protein with four transmembrane domains (TM), one small extracellular domain, and one large extracellular domain. Homo sapiens or human CD37 protein is encoded by a nucleic acid sequence that encodes the amino acid sequence shown in SEQ ID NO: 62 (human CD37 protein: UniprotKB/Swissprot P11049). In this amino acid sequence, residues 112-241 correspond to the large extracellular domain, residues 39-59 correspond to the small extracellular domain, and the remaining residues correspond to the transmembrane and cytoplasmic domains. Macaca fascicularis or cynomolgus monkey CD37 protein is encoded by a nucleic acid sequence that encodes the amino acid sequence set forth in SEQ ID NO: 63 (cynomolgus monkey CD37 protein; Genbank Accession No. XP_005589942). Unless contradicted by context, the term "CD37" refers to "human CD37." The term "CD37" includes any variants, isoforms, and orthologues of CD37 that are naturally expressed by cells, including tumor cells, or expressed on cells transfected with the CD37 gene or cDNA.

The term "human CD20" or "CD20" refers to human CD20 (UniProtKB/Swiss-Prot No P11836) and includes any variants, isoforms, and orthologues of CD20 naturally expressed by cells, including tumor cells, or expressed on cells transfected with the CD20 gene or cDNA. Orthologues include rhesus monkey CD20 (macaca mulatta; UniProtKB/Swiss-Prot No H9YXP1) and cynomolgus monkey CD20 (Macaca fascicularis).

The terms "antibody that binds to CD37," "anti-CD37 antibody," "CD37-binding antibody," "CD37-specific antibody," and "CD37 antibody," which may be used interchangeably herein, refer to any antibody that binds to an epitope on the extracellular portion of CD37.

The term "antibody (Ab)" in the context of the present invention refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or any derivative thereof, that has the ability to specifically bind to an antigen under typical physiological conditions with a half-life of a significant period, e.g., at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3 days, 4 days, 5 days, 6 days, 7 days, or more days, or any other relevant functionally determined period (e.g., a period sufficient to induce, promote, enhance, and/or modulate a physiological response associated with the binding of the antibody to the antigen and/or a period sufficient for the antibody to mobilize an effector activity). The variable regions of the heavy and light chains of an immunoglobulin molecule contain the binding domains that interact with the antigen. The constant region of antibody (Ab) can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system, such as C1q (the first component in the classical pathway of complement activation).As indicated above, the term "antibody" as used herein includes fragments of antibodies that are antigen-binding fragments, i.e., retain the ability to specifically bind to antigen, unless otherwise stated or clearly contradicted by the context.It has been shown that the antigen-binding function of antibody can be exerted by fragments of full-length antibodies. Examples of antigen-binding fragments encompassed by the term "antibody" are: (i) a Fab' or Fab fragment, which is a monovalent fragment consisting of the _VL , _VH , _CL , and _CHI domains, or a monovalent antibody as described in WO2007059782 (Genmab); (ii) a F(ab') ₂ fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment, which consists essentially of the _VH and _CHI domains; (iv) an Fv fragment, which consists essentially of the _VL and _VH domains of a single arm of an antibody; (v) a dAb fragment, which consists essentially of the _VH domain, also called a domain antibody (Holt et al; Trends Biotechnol. 2003 Nov; 21 (11):484-90) (Ward et al., Nature 341 , 544-546 (1989)); (vi) a camelid or nanobody (Revets et al; Expert Opin Biol Ther. 2005 Jan; 5 (1):111-24) and (vii) isolated complementarity determining regions (CDRs). Furthermore, although the two domains _VL and _VH of the Fv fragment are encoded by separate genes, they may be linked by a synthetic linker that allows them to be made as a single protein chain ( _{the VL} and _VH domains pair to form a monovalent molecule) using recombinant techniques (known as single chain antibodies or single chain Fvs (scFvs). See, for example, Bird et al., Science 242 , 423-426 (1988) and Huston et al., PNAS USA 85 , 5879-5883 (1988)). Such single chain antibodies are encompassed by the term "antibody" unless otherwise stated or clearly indicated by the context. Although such fragments are generally included in the meaning of antibodies, they collectively and each independently are unique features of the present invention and exhibit various biological properties and usefulness. These and other useful antibody fragments and bispecific formats of such fragments in the context of the present invention are further described herein. In the case of bispecific antibodies contained in the pharmaceutical compositions of the present invention, such fragments are linked to the Fc domain. The term "antibody" should also be understood to include polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, as well as antibody fragments (antigen-binding fragments) that retain the ability to specifically bind to an antigen, provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques, unless otherwise specified. The antibody generated can have any isotype.

The term "bispecific antibody" refers to an antibody having specificity for at least two different, generally non-overlapping epitopes. Such epitopes may be on the same target or on different targets. In the context of the present invention, the epitopes are on the same target (i.e., CD37). Examples of different classes of bispecific antibodies that contain an Fc region include, but are not limited to, asymmetric bispecific molecules, e.g., IgG-like molecules with complementary CH3 domains; and symmetric bispecific molecules, e.g., recombinant IgG-like dual targeting molecules, in which each antigen-binding region of the molecule binds to at least two different epitopes.

Examples of bispecific molecules include Triomab® (Trion Pharma/Fresenius Biotech, WO/2002/020039), Knobs-into-Holes (Genentech, WO1998/50431), CrossMAbs (Roche, WO2009/080251, WO2009/080252, WO2009/080253), electrostatically matched Fc heterodimer molecules (Amgen, EP1870459 and WO2009089004; Chugai, US201000155133; Oncomed, WO2010/129304), LUZ-Y (Genentech), DIG-body, PIG-body and TIG-body (Pharmabcine), Strand Exchange Engineered Domain body (SEEDbody) (EMD Serono, WO2007110205), bispecific IgG1 and IgG2 (Pfizer/Rinat, WO2011/143545), Azymetric scaffolds (Zymeworks/Merck, WO2012058768), mAb-Fv (Xencor, WO2011/028952), XmAb (Xencor), bivalent bispecific antibodies (Roche, WO20 09/080254), bispecific IgG (Eli Lilly), DuoBody® molecule (Genmab A/S, WO2011/131746), DuetMab (Medimmune, US2014/0348839), Biclonics (Merus, WO2013/157953), NovImmune (κλBodies, WO2012/023053), FcΔAdp (Regeneron, WO2010/151792), (DT)-Ig (GSK/Domantis), Two-in-one Antibody or Dual Action Fabs (Genentech, Adimab), mAb2 (F-Star, WO2008/003116), Zybody™ molecules (Zygenia), CovX-body (CovX/Pfizer), FynomAbs (Covagen/Janssen Cilag), DutaMab (Dutalys/Roche), iMab (MedImm une), Dual Variable Domain (DVD)-IgTM (Abbott), Dual Domain Double Head Antibody (Unilever; Sanofi Aventis, WO2010/0226923), Ts2Ab (MedImmune/AZ), BsAb (Zymogenetics), HERCULES (Biogen Idec, US7,951,918), scFv fusions (Genentech/Roche, Novartis, Immunomedics, Changzhou Adam Biotech Inc, CN102250246), TvAb (Roche, WO2012/025525, WO2012/025530), ScFv/Fc fusions, SCORPION (Emergent BioSolutions/Trubion, Zymogenetics/BMS), Interceptor (Emergent), Dual Affinity Retargeting These include, but are not limited to, Fc-DART™ Technology (MacroGenics, WO2008/157379, WO2010/080538), BEAT (Glenmark), Di-Diabody (Imclone/Eli Lilly) and chemically cross-linked mAbs (Karmanos Cancer Center) and covalently fused mAbs (AIMM therapeutics).

As used herein, the term "full-length antibody" refers to an antibody (e.g., a parent or variant antibody) that contains all the heavy and light chain constant and variable domains that correspond to those normally found in a wild-type antibody of that class or isotype.

The term "chimeric antibody" as used herein refers to an antibody whose variable region is derived from a non-human species (e.g., rodent) and whose constant region is derived from a different species (e.g., human). Chimeric antibodies can be produced by antibody engineering. "Antibody engineering" is a term used collectively for various types of antibody modifications, a process well known to those skilled in the art. In particular, chimeric antibodies can be produced by using standard DNA techniques such as those described in Sambrook et al., 1989, Molecular Cloning: A laboratory Manual, New York: Cold Spring Harbor Laboratory Press, Ch. 15. Thus, chimeric antibodies can be genetically or enzymatically modified recombinant antibodies. Producing chimeric antibodies is within the knowledge of those skilled in the art, and thus, the production of chimeric antibodies may be performed by methods other than those described herein. Chimeric monoclonal antibodies for therapeutic use have been developed to reduce the immunogenicity of the antibody. Such antibodies generally may contain a non-human (e.g., mouse) variable region specific for an antigen of interest and human constant antibody heavy and light chain domains. The term "variable region" or "variable domain" as used with respect to chimeric antibodies refers to the region that contains the CDR and framework regions of both the heavy and light immunoglobulin chains.

As used herein, the term "oligomer" refers to a molecule that is composed of more than one, but limited, number of monomeric units (e.g., an antibody), as opposed to a polymer that is composed of, at least in principle, an infinite number of monomers. Exemplary oligomers are dimers, trimers, tetramers, pentamers, and hexamers. Similarly, as used herein, "oligomerization," e.g., "hexamerization," refers to the increased partitioning of antibodies and/or other dimeric proteins that contain a target binding region into oligomers, e.g., hexamers. The increased formation of oligomers, such as hexamers, is due to increased Fc-Fc interactions following binding to membrane-bound targets.

As used herein, the terms "antigen-binding region", "binding region", or "antigen-binding domain" refer to the region of an antibody capable of binding to an antigen. This binding region is generally defined by the VH and VL domains of the antibody, which may be further subdivided into regions of hypervariability (or hypervariable regions that may be hypervariable in the sequence and/or shape of structurally defined loops), also called complementarity determining regions (CDRs), interspersed between more conserved regions called framework regions (FRs). An antigen can be any molecule, e.g., a polypeptide, present, for example, on a cell, on a bacterium, or on a virion, or in solution. The terms "antigen" and "target" may be used interchangeably with respect to the present invention, unless the context is inconsistent.

As used herein, the term "target" refers to a molecule to which the antigen-binding region of an antibody binds. Targets include any antigen against which an antibody is generated. The terms "antigen" and "target" are used interchangeably with respect to antibodies and may have the same meaning and intent with respect to any aspect or embodiment of the present invention.

The term "humanized antibody" as used herein refers to a genetically engineered non-human antibody containing a human antibody constant domain and a non-human variable domain that has been modified to contain a high level of sequence homology to the human variable domain. This can be achieved by grafting the six non-human antibody complementarity determining regions (CDRs) that together form the antigen binding site into a homologous human acceptor framework region (FR) (see W092/22653 and EP0629240). Substitution of framework residues from the parent antibody (i.e., non-human antibody) into human framework regions (backmutations) may be required to fully reconstitute the binding affinity and specificity of the parent antibody. Structural homology modeling can aid in the identification of amino acid residues in the framework regions that are important for antibody binding. Thus, a humanized antibody may contain primarily human framework regions, including non-human CDR sequences and, optionally, one or more amino acid backmutations to non-human amino acid sequences, and a fully human constant region. Optionally, further amino acid modifications, not necessarily back mutations, may be applied to obtain a humanized antibody with favorable properties, such as affinity and biochemical properties.

Humanized antibodies can be generated by immunizing rabbits, humanizing the rabbit antibodies using germline humanization (CDR grafting) techniques, and, if necessary, backmutating residues that may be critical for antibody binding (identified in structural modeling) to rabbit residues. Screening for potential T-cell epitopes can be applied.

The term "human antibody" as used herein refers to an antibody having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, e.g., a mouse, have been grafted onto human framework sequences. Human monoclonal antibodies can be produced by a variety of techniques, including conventional monoclonal antibody techniques, e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256: 495 (1975). Although somatic cell hybridization procedures are preferred, in principle other techniques for producing monoclonal antibodies can also be used, e.g., viral or oncogenic transformation of B lymphocytes or phage display techniques using libraries of human antibody genes.

A suitable animal system for preparing hybridomas secreting human monoclonal antibodies is the murine system. Hybridoma production in the mouse is a very well established procedure. Immunization protocols and techniques for isolation of immunized spleen cells for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.

Human monoclonal antibodies can be produced, for example, using transgenic or transchromosomal mice or rabbits that have parts of the human immune system rather than the mouse or rabbit system.

The term "immunoglobulin" refers to a class of structurally related glycoproteins that consist of two pairs of polypeptide chains, a pair of low molecular weight light (L) chains and a pair of heavy (H) chains, all four chains interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, for example, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)). Briefly, each heavy chain generally consists of a heavy chain variable region (abbreviated herein as _VH or VH) and a heavy chain constant region (abbreviated herein as _CH or CH). The heavy chain constant region generally consists of three domains, _CH1 , _CH2 , and _CH3 . Each light chain generally consists of a light chain variable region (abbreviated herein as _VL or VL) and a light chain constant region (abbreviated herein as _CL or CL). The light chain constant region generally consists of one domain, _CL . The _VH and _VL regions can be further subdivided into regions of hypervariability, also called complementarity determining regions (CDRs) (or hypervariable regions that may be hypervariable in the sequence and/or configuration of structurally defined loops), interspersed between more conserved regions, called framework regions (FRs). Each _VH and _VL is generally composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196 , 901-917 (1987)). Unless otherwise stated or contradicted by the context, CDR sequences herein are identified according to the IMGT rules (Brochet X., Nucl Acids Res. 2008;36:W503-508 and Lefranc MP., Nucleic Acids Research 1999;27:209-212; see also the internet http address http://www.imgt.org/). Unless otherwise stated or contradicted by the context, references to amino acid positions in the constant regions of the present invention are according to EU numbering (Edelman et al., Proc Natl Acad Sci US A. 1969 May;63(1):78-85; Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition. 1991 NIH Publication No. 91-3242).

Unless contradicted by context, the term "Fab arm" or "arm" as used herein refers to one heavy-light chain pair and is used interchangeably with "half molecule" herein. Thus, a "Fab arm" includes the variable regions of the heavy and light chains and the constant region of the light chain and the constant region of the heavy chain, which includes the CH1, hinge, CH2 and CH3 regions of an immunoglobulin. "CH1 region" refers, for example, to the region of a human IgG1 antibody corresponding to amino acids 118-215 according to EU numbering. Thus, a Fab fragment includes the binding region of an immunoglobulin.

The terms "fragment crystallizable region", "Fc region", "Fc fragment" or "Fc domain", which may be used interchangeably herein, refer to an antibody region that includes at least the hinge region, CH2 domain and CH3 domain arranged from the amino terminus to the carboxy terminus. The Fc region of an IgG1 antibody can be generated, for example, by digestion of an IgG1 antibody with papain. The Fc region of an antibody can mediate binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system, such as C1q (the first component in the classical pathway of complement activation). The term "hinge region" as used herein is intended to refer to the hinge region of an immunoglobulin heavy chain. Thus, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216-230 according to EU numbering.

As used herein, the term "core hinge" or "core hinge region" refers to the four amino acids corresponding to positions 226-229 of a human IgG1 antibody.

The term "CH2 region" or "CH2 domain" as used herein is intended to refer to the CH2 region of an immunoglobulin heavy chain. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to EU numbering. However, the CH2 region can also be of any of the other isotypes or allotypes described herein.

As used herein, the term "CH3 region" or "CH3 domain" is intended to refer to the CH3 region of an immunoglobulin heavy chain. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to EU numbering. However, the CH3 region can also be of any of the other isotypes or allotypes described herein.

As used herein, the term "isotype" refers to the immunoglobulin class (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) encoded by heavy chain constant region genes.

In the context of the present invention, the term "monovalent antibody" refers to an antibody molecule that can only bind to a single antigen molecule and is therefore incapable of antigen cross-linking.

A "CD37 antibody" or "anti-CD37 antibody" is an antibody as described above that specifically binds to the antigen CD37.

A "CD37xCD37 antibody" or "anti-CD37xCD37 antibody" is a bispecific antibody that contains two different antigen-binding regions, one that specifically binds to a first epitope on the antigen CD37 and the other that specifically binds to a different epitope on CD37.

In one embodiment, the bispecific antibody included in the pharmaceutical composition of the invention is isolated. As used herein, "isolated bispecific antibody" is intended to refer to a bispecific antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated bispecific antibody that specifically binds to CD37 is substantially free of monospecific antibodies that specifically bind to CD37).

The term "epitope" refers to a protein determinant capable of binding to the antigen-binding region ("paratope") of an antibody. Epitopes usually consist of surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural features and specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. "Structural epitopes" or "functional epitopes" can be determined by epitope mapping techniques. Structural epitopes are defined as residues in the structure that are in direct contact with the antibody and can be evaluated, for example, by structure-based methods such as X-ray crystallography. Structural epitopes can include amino acid residues that are directly involved in antibody binding and other amino acid residues that are not directly involved in binding, for example amino acid residues that are effectively blocked or hidden by the antibody (in other words, the amino acid residues are in the footprint of the antibody). Functional epitopes are defined as residues that contribute energetically to antigen-antibody binding interactions and can be assessed, for example, by site-directed mutagenesis such as alanine scanning (Cunningham, B. C, & Wells, J. A. (1993) Journal of Molecular Biology; Clackson, T., & Wells, J. (1995) Science, 267(5196), 383-386). Functional epitopes can include amino acid residues that are directly involved in antibody binding and other amino acid residues that are not directly involved in binding, for example, amino acid residues that cause conformational changes to the positions of residues involved in direct interactions (Greenspan, N. S., & Di Cera, E. (1999) Nature Biotechnology, 17(10), 936-937). In the case of antigen-antibody interactions, functional epitopes can be used to distinguish antibody molecules from each other. Functional epitopes can be determined using the alanine scanning method described in Example 17. Thus, amino acids in a protein can be substituted with alanine to generate a series of mutant proteins, which have reduced binding of the antigen-binding region of an antibody to the mutant proteins compared to the wild-type protein; reduced binding is determined as a normalized log(fold change) in binding of the antibody (expressed as a z-score) of less than -1.5, as described in Example 17.

As used herein, the term "monoclonal antibody" refers to a preparation of antibody molecules that are essentially uniform in molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody exhibiting a single binding specificity having variable and constant regions derived from human germline immunoglobulin sequences. Human monoclonal antibodies can be produced by hybridomas, which include B cells obtained from a transgenic or transchromosomal non-human animal, e.g., a transgenic mouse, whose genome includes human heavy and light chain transgenes, fused to an immortalized cell.

As used herein, the term "binding" in reference to the binding of an antibody to a predetermined antigen generally refers to binding with an affinity corresponding to a K D of about 10 ⁻⁶ M or less, such as about 10 −7 M or less, such as about 10 ⁻⁸ M or less, such as about 10 ⁻⁹ M or less, about 10 ⁻¹⁰ M or less, or about 10 −11 M or less, e.g., as determined by BioLayer Interferometry (BLI) technology on an Octet HTX instrument using the antibody as the ligand and the antigen as the analyte, and the antibody binds to the predetermined antigen with an affinity corresponding to a K _D that is at least ¹⁰ -fold lower, such as at least 100 ^- fold lower, such as at least 1,000-fold lower, such as at least 10,000-fold lower, such as at least 100,000-fold lower, for example at least 100,000-fold lower, than the K _D for binding to a non-specific antigen other than the predetermined antigen or a closely related antigen (e.g., BSA, casein ₎ . How low the _KD of binding is depends on the _KD of the antibody, thus, if the _KD of an antibody is very low, the _KD of binding to an antigen may be at least 10,000 times lower than the _KD of binding to a non-specific antigen (i.e., the antibody is highly specific).

The term "K _D " (M) as used herein refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.

As used herein, "affinity" and " _KD " are inversely related, i.e., a higher affinity is intended to refer to a lower _KD and a lower affinity is intended to refer to a higher _KD .

As used herein, an antibody that "competes" or "cross-competes" with another antibody (i.e., a reference antibody) is used interchangeably with an antibody that "blocks" or "cross-blocks," and means that the antibody and the reference antibody compete for binding to human CD37, e.g., as determined in the assay described in Example 7 herein. In one embodiment, the antibody binds at less than 50%, e.g., less than 20%, e.g., less than 15%, of its maximal binding in the presence of the competing reference antibody.

As used herein, an antibody that "does not compete" or "cross-compete" or "does not block" another antibody (i.e., a reference antibody) means that the antibody and the reference antibody do not compete for binding to human CD37 as determined in the assay described in Example 7 herein. For some pairs of antibodies and reference antibodies, non-competition in the assay of Example 7 is observed only when one antibody is binding to an antigen on a cell and the other antibody is used to compete, and not vice versa. The terms "does not compete with" or "non-competing" or "non-blocking" as used herein are also intended to encompass such combinations of antibodies. In one embodiment, the antibody binds at least 75%, e.g., at least 80%, e.g., at least 85%, of its maximum binding in the presence of the reference antibody.

As used herein, the term "Fc-Fc interaction enhancing mutation" refers to a mutation in an IgG antibody that enhances the Fc-Fc interaction between adjacent IgG antibodies that are bound to a cell surface target. This can result in enhanced oligomerization (e.g., hexamerization) of the antibody upon binding to a target, although the antibody molecule remains monomeric in solution, as described in WO2013/004842 and WO2014/108198, both of which are incorporated herein by reference.

As used herein, the term "Fc effector function" or "Fc-mediated effector function" is intended to refer to a function that is a result of the binding of a polypeptide or antibody to its target (e.g., an antigen) on a cell membrane and the subsequent interaction of the IgG Fc domain with molecules of the innate immune system (e.g., soluble or membrane-bound molecules). Examples of Fc effector functions include (i) C1q binding, (ii) complement activation, (iii) complement-dependent cytotoxicity (CDC), (iv) antibody-dependent cellular cytotoxicity (ADCC), (v) Fc-gamma receptor binding, (vi) antibody-dependent cellular phagocytosis (ADCP), (vii) complement-dependent cytotoxicity (CDCC), (viii) complement-enhanced cytotoxicity, (ix) antibody-mediated opsonization (binding of an antibody to a complement receptor), (x) opsonization, and (xi) any combination of (i)-(x).

As used herein, the term "heterodimeric interaction between the first and second CH3 regions" refers to the interaction between the first CH3 region of the first Fc region and the second CH3 region of the second Fc region in a first CH3/second CH3 heterodimeric protein. Bispecific antibodies are an example of heterodimeric proteins.

As used herein, the term "homodimeric interaction of a first and second CH3 region" refers to an interaction between a first CH3 region and another first CH3 region in a first CH3/first CH3 homodimeric protein, and an interaction between a second CH3 region and another second CH3 region in a second CH3/second CH3 homodimeric protein. A monoclonal antibody is an example of a homodimeric protein.

The term "reducing conditions" or "reducing environment" refers to conditions or circumstances in which a substrate, such as a cysteine residue in the hinge region of an antibody, is more likely to be reduced than oxidized.

The present invention also provides pharmaceutical compositions comprising bispecific antibodies that are functional variants of the _VL or _VH regions of the bispecific antibodies of the examples. Functional variants of _VL , _VH , or CDRs used in the context of bispecific antibodies still allow each arm of the bispecific antibody to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% or more) of the affinity and/or specificity/selectivity of the parent bispecific antibody, and in some cases such bispecific antibodies may be associated with higher affinity, selectivity, and/or specificity than the parent bispecific antibody. Such functional variants generally retain significant sequence identity with the parent bispecific antibody. The percent identity between two sequences is a function of the number of identical positions shared by both sequences (i.e., percent homology = number of identical positions/total number of positions x 100), taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. The percent identity between two nucleotide or amino acid sequences can be determined, for example, using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988), as incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970).

Exemplary variants include variants that differ from the VH and/or VL and/or CDR regions of the parent bispecific antibody sequence primarily by conservative substitutions. For example, 10, e.g., 9, 8, 7, 6, 5, 4, 3, 2, or 1 of the substitutions in the variant are conservative amino acid residue substitutions. Preferably, the variant contains at most 10 amino acid substitutions, e.g., at most 9, 8, 7, 6, 5, 4, 3, 2, or at most 1 amino acid substitution, in the VH and/or VL regions of the parent antibody. Preferably, such substitutions are conservative substitutions, especially when in the CDR sequences.

For the purposes of the present invention, conservative substitutions may be defined as substitutions within the amino acid classes set forth in the table below.

Conservatively substituted amino acid residue classes

In the context of the present invention, unless otherwise indicated, the following notations are used to describe mutations: i) substitution of an amino acid at a given position is noted, for example, K409R (meaning substitution of lysine at position 409 with arginine); ii) in the case of a particular variant, a designated three- or one-letter code is used to indicate any amino acid residue, for example the codes Xaa and X. For example, substitution of lysine at position 409 with arginine is designated K409R; substitution of lysine at position 409 with any amino acid residue is designated K409X. In case of deletion of lysine at position 409, it is indicated by K409*.

As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell into which an expression vector has been introduced, such as an expression vector encoding an antibody used in the present invention. Recombinant host cells include, for example, transfectomas, such as CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C6, or NS0 cells, and lymphocytic cells.

The term "treatment" refers to the administration of an effective amount of a pharmaceutical composition of the present invention with the intent to alleviate, ameliorate, inhibit, or eradicate (cure) a symptom or disease state.

The term "effective amount" or "therapeutically effective amount" refers to an amount effective to achieve a desired therapeutic result at the dosage and duration required. A therapeutically effective amount of a bispecific antibody may vary according to factors such as the disease state, the age, sex, and weight of the individual, and the ability of the bispecific antibody to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or adverse effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects.

As described above, in a main aspect, the present invention provides a method for producing a method for the treatment of a pulmonary arthritis, comprising:
a) bispecific antibodies;
b) histidine buffer;
c) 50-300 mM sugar and/or 50-300 mM polyol, and
d) 0.01 to 0.1% polysorbate 80
A pharmaceutical composition comprising:
the pH of the composition is between 4.5 and 6.8; and the bispecific antibody comprises first and second antigen-binding regions that bind human CD37 having the sequence of SEQ ID NO: 62, and first and second Fc regions of a human immunoglobulin, the first and second Fc regions comprising one or more amino acid mutations that enhance Fc-Fc interactions between the bispecific antibodies upon binding to a membrane-bound target compared to Fc-Fc interactions between bispecific antibodies not comprising the mutations, and the first antigen-binding region comprises the CDR sequence:
VH CDR1 sequence as set forth in SEQ ID NO: 16;
VH CDR2 sequence as set forth in SEQ ID NO: 17;
VH CDR3 sequence as set forth in SEQ ID NO: 18;
The VL CDR1 sequence according to SEQ ID NO: 20;
a VL CDR2 sequence that is a KAS; and
The second antigen-binding region comprises a VL CDR3 sequence as set forth in SEQ ID NO: 21, and the second antigen-binding region comprises the CDR sequence:
VH CDR1 sequence as set forth in SEQ ID NO: 23;
VH CDR2 sequence as set forth in SEQ ID NO: 24;
VH CDR3 sequence as set forth in SEQ ID NO: 25;
The VL CDR1 sequence set forth in SEQ ID NO: 27;
a VL CDR2 sequence which is YAS, and
The pharmaceutical composition comprises the VL CDR3 sequence set forth in SEQ ID NO: 31.

The bispecific anti-CD37 antibody contained in the pharmaceutical composition of the present invention binds to two different epitopes on CD37. The two epitopes are such that both binding arms can bind to the same protein molecule and thus each binding arm does not block the binding of the other arm of the bispecific molecule and/or does not compete with the other binding arm of the bispecific molecule for binding. The bispecific antibody also contains a mutation that enhances the Fc-Fc interaction between two or more bispecific antibody molecules. This has the effect that the bispecific molecule forms an oligomer upon binding to CD37 expressed on the cell membrane of the target cell. The Fc-Fc interaction is enhanced compared to a molecule that is identical except for the mutation. Preferably, the mutation is in the Fc region of the bispecific molecule. In one embodiment, it is a single amino acid substitution in the Fc region of the bispecific molecule. It is preferably a symmetric substitution, which means that both half molecules (parent antibodies) have the mutation. A further advantage of the bispecific antibody is that it has enhanced CDC and/or ADCC effector function compared to the same bispecific molecule without the Fc-Fc interaction enhancing mutation. Surprisingly, the bispecific molecule also has improved CDC and/or ADCC compared to the combination of two parent monoclonal anti-CD37 antibodies mutated to have enhanced Fc-Fc interactions, and also compared to either parent monoclonal anti-CD37 antibody alone mutated to have enhanced Fc-Fc interactions. Thus, the bispecific antibody is more potent in inducing CDC and/or ADCC than a combination of an antibody having a first antigen binding region and a second antibody having a second antigen binding region, both antibodies containing Fc-Fc interaction enhancing mutations, or compared to a single monoclonal anti-CD37 antibody having either the first or second antigen binding region and containing Fc-Fc interaction enhancing mutations.

The present invention also provides a stable pharmaceutical composition comprising an antibody having a binding arm that binds to CD37.

Thus, in one aspect, the present invention provides a method for producing a composition comprising:
i) antibodies,
j) histidine buffer;
k) 50-300 mM sugar and/or 50-300 mM polyol, and
l) 0.01-0.1% polysorbate 80
A pharmaceutical composition comprising:
the antibody comprises a first antigen-binding region that binds human CD37 having the sequence of SEQ ID NO: 62, and a first and a second Fc region of a human immunoglobulin, the first and second Fc regions comprising one or more amino acid mutations that enhance Fc-Fc interactions between the antibodies upon binding to a membrane-bound target compared to Fc-Fc interactions between bispecific antibodies not having the mutations, the first antigen-binding region comprising the CDR sequence:
VH CDR1 sequence as set forth in SEQ ID NO: 16;
VH CDR2 sequence as set forth in SEQ ID NO: 17;
VH CDR3 sequence as set forth in SEQ ID NO: 18;
The VL CDR1 sequence according to SEQ ID NO: 20;
a VL CDR2 sequence that is a KAS, and
The pharmaceutical composition comprises the VL CDR3 sequence set forth in SEQ ID NO: 21.

In a further aspect, the present invention provides a method for producing a method for treating a cancer cell comprising the steps of:
m) antibodies,
n) histidine buffer;
o) 50-300 mM sugar and/or 50-300 mM polyol, and
p) 0.01 to 0.1% polysorbate 80
A pharmaceutical composition comprising:
the antibody comprises a second antigen-binding region that binds human CD37 having the sequence of SEQ ID NO: 62, and a first and a second Fc region of a human immunoglobulin, the first and second Fc regions comprising one or more amino acid mutations that enhance Fc-Fc interactions between the antibodies upon binding to a membrane-bound target compared to Fc-Fc interactions between bispecific antibodies not having the mutations, the second antigen-binding region comprising the CDR sequence:
VH CDR1 sequence as set forth in SEQ ID NO: 23;
VH CDR2 sequence as set forth in SEQ ID NO: 24;
VH CDR3 sequence as set forth in SEQ ID NO: 25;
The VL CDR1 sequence set forth in SEQ ID NO: 27;
a VL CDR2 sequence which is YAS, and
The pharmaceutical composition comprises the VL CDR3 sequence set forth in SEQ ID NO: 31.

In one embodiment, the pharmaceutical composition of the invention comprises 5-100 mg/mL of the bispecific antibody, such as 10-50 mg/mL of the bispecific antibody, such as 10-30 mg/mL of the bispecific antibody, such as 20 mg/mL of the bispecific antibody.

In one embodiment, the pharmaceutical composition of the invention comprises 5-100 mg/mL of antibody, e.g., 10-50 mg/mL of antibody, e.g., 10-30 mg/mL of antibody, e.g., 20 mg/mL of antibody.

In one embodiment, the pharmaceutical composition of the invention comprises 10-100 mM histidine, such as 10-50 mM histidine, such as 10-30 mM histidine, such as 20 mM histidine. In one embodiment, the histidine is histidine-HCl.

In one embodiment, the pharmaceutical composition of the invention comprises a sugar such as sucrose or trehalose. In one embodiment, the sugar is sucrose and the pharmaceutical composition comprises 75-275 mM sucrose, such as 100-250 mM, such as 100 mM sucrose or 250 mM sucrose. In a further embodiment thereof, the pharmaceutical composition does not comprise a polyol.

In another embodiment, the pharmaceutical composition of the invention comprises a polyol which is sorbitol or mannitol, the pharmaceutical composition preferably comprising 75-275 mM sorbitol or 75-275 mM mannitol, such as 100-250 mM sorbitol or 100-250 mM mannitol, such as 100 mM sorbitol or 100 mM mannitol or 250 mM sorbitol or 100 mM mannitol. In a further embodiment thereof, the pharmaceutical composition does not comprise a sugar.

In one embodiment, the pharmaceutical composition of the invention comprises 0.01-0.05% polysorbate 80 (Tween 80), e.g., 0.01%-0.04% polysorbate 80, e.g., 0.02% polysorbate 80 or 0.04% polysorbate 80.

In one embodiment, the pharmaceutical composition of the present invention has a pH of 5.5 to 6.5, for example 5.5 or 6.5.

In one embodiment, the pharmaceutical composition of the invention has a pH of 5.5 to 6.5, such as 5.6 to 6.4 or 5.7 to 6.3, for example 5.8 to 6.2, such as 5.9 to 6.1.

In one embodiment, the pharmaceutical composition of the present invention has a pH of about 6.

In one embodiment, the pharmaceutical composition of the present invention, the composition further comprises sodium chloride, for example 25 to 250 mM sodium chloride, for example 100 to 150 mM sodium chloride, for example 100 mM or 150 mM sodium chloride.

In one embodiment, the pharmaceutical composition of the invention further comprises arginine, e.g., 25-200 mM arginine, e.g., 50-100 mM arginine, e.g., 75 mM arginine. In one embodiment, the arginine is arginine-HCl.

In one embodiment, the pharmaceutical composition of the present invention comprises:
a) 20 mg/mL bispecific antibody, 20 mM histidine, 250 mM sucrose, and 0.02% or 0.04% polysorbate 80 at pH 5.5-6.5; or
b) 20 mg/mL of bispecific antibody, 20 mM histidine, 100 mM sucrose, 0.02% or 0.04% polysorbate 80, and 100-150 mM, preferably 100 mM, sodium chloride at pH 5.5-6.5; or
c) 20 mg/mL of bispecific antibody, 20 mM histidine, 100 mM sucrose, 0.02% or 0.04% polysorbate 80, 75 mM arginine, and 100-150 mM, preferably 100 mM, sodium chloride at pH 5.5-6.5; or
d) 20 mg/mL bispecific antibody, 20 mM histidine, 100 mM sucrose, 0.02% or 0.04% polysorbate 80, and 75 mM arginine, at pH 5.5-6.5.

In a further embodiment, the pharmaceutical composition of the invention consists of the following components in aqueous solution:
a) 20 mg/mL bispecific antibody, 20 mM histidine, 250 mM sucrose, and 0.02% or 0.04% polysorbate 80 at pH 5.5-6.5; or
b) 20 mg/mL bispecific antibody, 20 mM histidine, 100 mM sucrose, 0.02% or 0.04% polysorbate 80, and 100-150 mM, preferably 100 mM, sodium chloride at pH 5.5-6.5; or
c) 20 mg/mL bispecific antibody, 20 mM histidine, 100 mM sucrose, 0.02% or 0.04% polysorbate 80, 75 mM arginine, and 100-150 mM, preferably 100 mM, sodium chloride at pH 5.5-6.5; or
d) 20 mg/mL bispecific antibody, 20 mM histidine, 100 mM sucrose, 0.02% or 0.04% polysorbate 80, and 75 mM arginine at pH 5.5-6.5.

In a further embodiment of the invention, the first antigen-binding region of the bispecific antibody has the following VH and VL sequences:
i) the VH sequence set forth in SEQ ID NO: 15 and the VL sequence set forth in SEQ ID NO: 19, or
ii) the sequences of the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of the first antigen-binding region are
VH CDR1 sequence as set forth in SEQ ID NO: 16;
VH CDR2 sequence as set forth in SEQ ID NO: 17;
VH CDR3 sequence as set forth in SEQ ID NO: 18;
The VL CDR1 sequence according to SEQ ID NO: 20;
a VL CDR2 sequence that is a KAS, and
Provided that the VL CDR3 sequence as set forth in SEQ ID NO:21 remains the VH sequence having at least 90% identity, such as at least 95% identity, such as at least 98% identity, such as at least 99% identity, and the VL sequence having at least 90% identity, such as at least 95% identity, such as at least 98% identity, such as at least 99% identity, to the VH and VL sequences of SEQ ID NO:15 and 19,

In another embodiment of the invention, the first antigen-binding region of the bispecific antibody has the following VH and VL sequences:
iii) the VH sequence set forth in SEQ ID NO: 15 and the VL sequence set forth in SEQ ID NO: 127; or
iv) the sequences of the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of the first antigen-binding region are
VH CDR1 sequence as set forth in SEQ ID NO: 16;
VH CDR2 sequence as set forth in SEQ ID NO: 17;
VH CDR3 sequence as set forth in SEQ ID NO: 18;
The VL CDR1 sequence according to SEQ ID NO: 20;
a VL CDR2 sequence that is a KAS, and
Provided that the VL CDR3 sequence as set forth in SEQ ID NO:21 remains the VH sequence having at least 90% identity, such as at least 95% identity, such as at least 98% identity, such as at least 99% identity, and the VL sequence having at least 90% identity, such as at least 95% identity, such as at least 98% identity, such as at least 99% identity, to the VH and VL sequences of SEQ ID NO:15 and 19,

In a further embodiment of the invention, the second antigen-binding region of the bispecific antibody comprises
(i) the VH sequence set forth in SEQ ID NO: 22, and the VL sequence set forth in SEQ ID NO: 29; or (ii) the sequences of the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of the second antigen-binding region are
VH CDR1 sequence as set forth in SEQ ID NO: 23;
VH CDR2 sequence as set forth in SEQ ID NO: 24;
VH CDR3 sequence as set forth in SEQ ID NO: 25;
The VL CDR1 sequence set forth in SEQ ID NO: 27;
a VL CDR2 sequence which is YAS, and
The present invention also includes VH and VL sequences selected from the group comprising VH sequences having at least 90% identity, such as at least 95% identity, such as at least 98% identity, such as at least 99% identity, and VL sequences having at least 90% identity, such as at least 95% identity, such as at least 98% identity, such as at least 99% identity, to the VH and VL sequences of SEQ ID NOs: 22 and 29, provided that the VL CDR3 sequence as set forth in SEQ ID NO: 31 remains the same.

Thus, in another embodiment of the invention, a bispecific antibody comprises a first and a second antigen-binding region, the first antigen-binding region of the bispecific antibody comprises the VH and VL sequences of antibody 010 (i.e., SEQ ID NOs: 15 and 19), and the second antigen-binding region of the bispecific antibody comprises the VH and VL sequences of antibody 016 (i.e., SEQ ID NOs: 22 and 29).

In one embodiment of the invention, a bispecific antibody comprises a first and a second antigen-binding region, the first antigen-binding region of the bispecific antibody comprises the VH and VL sequences of antibody 010 (i.e., SEQ ID NOs: 15 and 127), and the second antigen-binding region of the bispecific antibody comprises the VH and VL sequences of antibody 016 (i.e., SEQ ID NOs: 22 and 29).

In a preferred embodiment of the invention, the bispecific antibody comprises a first and a second antigen-binding region, the first antigen-binding region of the bispecific antibody comprises the VH and VL sequences set forth in SEQ ID NOs: 15 and 127, respectively, and the second antigen-binding region of the bispecific antibody comprises the VH and VL sequences set forth in SEQ ID NOs: 22 and 29, respectively.

In one embodiment, the first antigen-binding region of the bispecific antibody has a functional epitope comprising one or more of the amino acids Y182, D189, T191, I192, D194, K195, V196, I197, and P199 of SEQ ID NO: 62 (CD37). In another embodiment, the first antigen-binding region binds to a functional epitope comprising one or more of the amino acids selected from the group consisting of Y182, D189, T191, I192, D194, K195, V196, I197, and P199 of SEQ ID NO: 62 (CD37). In a further embodiment, the first antigen-binding region of the bispecific antibody binds to a functional epitope on CD37, and binding to a mutant CD37 in which any one or more of the amino acid residues at positions corresponding to positions Y182, D189, T191, I192, D194, K195, V196, I197, and P199 of SEQ ID NO: 62 (CD37) are substituted with alanine is reduced compared to wild-type CD37 having the amino acid sequence set forth in SEQ ID NO: 62; the reduced binding is determined as a z-score (fold change) in binding of the antibody being less than -1.5, and the z-score (fold change) in binding is calculated as described in Example 17.

In one embodiment of the invention, the second antigen-binding region of the bispecific antibody has a functional epitope comprising one or more of the amino acids E124, F162, Q163, V164, L165, and H175 of SEQ ID NO: 62 (CD37). In another embodiment, the second antigen-binding region binds to a functional epitope comprising one or more of the amino acids selected from the group consisting of E124, F162, Q163, V164, L165, and H175 of SEQ ID NO: 62 (CD37). In a further embodiment, the second antigen-binding region of the bispecific antibody binds to a functional epitope on CD37 and has reduced binding to a mutant CD37 in which any one or more of the amino acid residues at positions corresponding to positions E124, F162, Q163, V164, L165, and H175 of SEQ ID NO: 62 (CD37) are substituted with alanine compared to wild-type CD37 having the amino acid sequence set forth in SEQ ID NO: 62; the reduced binding is determined as a z-score (fold change) in binding of the antibody being less than -1.5, and the z-score (fold change) in binding is calculated as described in Example 17.

Fc-Fc enhancing mutations In one embodiment of the present invention, one or more of the Fc-Fc interaction enhancing mutations in the first and second Fc regions of the bispecific antibody are amino acid substitutions. The Fc region of the bispecific antibody can be said to comprise two different Fc regions, one from each parent anti-CD37 antibody. The bispecific antibody can comprise one or more Fc-Fc interaction enhancing mutations in each half molecule. In one embodiment, the Fc-Fc interaction enhancing mutations are symmetric, i.e., the same mutations occur in the two Fc regions.

In one embodiment, the pharmaceutical composition of the invention comprises a bispecific antibody, wherein the one or more Fc-Fc interaction enhancing mutations in the first and second Fc regions are amino acid substitutions at one or more positions corresponding to amino acid positions 430, 440, and 345 in human IgG1 using the EU numbering system. In one embodiment, the pharmaceutical composition of the invention comprises a bispecific antibody, wherein the one or more Fc-Fc interaction enhancing mutations in the first and second Fc regions are amino acid substitutions at one or more positions corresponding to amino acid positions 430, 440, and 345 in human IgG1 using the EU numbering system, with the proviso that the substitution at 440 is 440Y or 440W.

In another embodiment, the pharmaceutical composition of the present invention comprises a bispecific antibody comprising at least one substitution in the first and second Fc regions selected from the group comprising E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y, and S440W. In a particularly preferred embodiment, the bispecific antibody comprises at least one substitution in the first and second Fc regions selected from E430G or E345K, preferably E430G. Hereby, a bispecific antibody is provided having enhanced Fc-Fc interactions between different antibodies having said mutations. It is believed that this mutation allows the formation of oligomers of antibodies on target cells, thereby enhancing CDC.

The Fc-Fc interaction enhancing mutations in the first and second Fc regions are preferably identical substitutions. Thus, in a preferred embodiment, the bispecific antibody has the same Fc-Fc interaction enhancing mutations in both Fc regions. The Fc region can also be described as Fc chain, and the antibody has two Fc chains that constitute the common Fc region of the antibody. Thus, in a preferred embodiment, each of the two Fc chains comprises a substitution at a position selected from the group of positions corresponding to amino acid positions 430, 440, and 345 when using the EU numbering system in human IgG1. In one embodiment, each of the two Fc chains comprises an E430G substitution, such that the bispecific antibody comprises two E430G substitutions. In another embodiment, each of the two Fc chains comprises an E345K substitution, such that the bispecific antibody comprises two E345K substitutions.

In an embodiment of the invention, the bispecific antibody is of the IgG1 isotype.

In an embodiment of the invention, the bispecific antibody is of the IgG2 isotype.

In an embodiment of the invention, the bispecific antibody is of the IgG3 isotype.

In an embodiment of the invention, the bispecific antibody is of the IgG4 isotype.

In an embodiment of the invention, the bispecific antibody is of the IgG isotype.

In one embodiment of the invention, the bispecific antibody is a combination of isotypes IgG1, IgG2, IgG3, and IgG4. For example, the first half antibody obtained from the first parent antibody may be of IgG1 isotype and the second half antibody obtained from the second parent antibody may be of IgG4 isotype, so that the bispecific antibody is a combination of IgG1 and IgG4. In another embodiment, the bispecific antibody is a combination of IgG1 and IgG2. In another embodiment, the bispecific antibody is a combination of IgG1 and IgG3. In another embodiment, the bispecific antibody is a combination of IgG2 and IgG3. In another embodiment, the bispecific antibody is a combination of IgG2 and IgG4. In another embodiment, the bispecific antibody is a combination of IgG3 and IgG4. Generally, the core hinge is an IgG1 type core hinge with the sequence CPPC, but can also be other stable hinges that do not allow the Fab arms to exchange in vivo (as is the case for IgG4 core hinges with the sequence CPSC).

In a preferred embodiment, the bispecific antibody is a full-length antibody.

In yet another embodiment of the invention, the bispecific antibody is a human antibody. In yet another embodiment of the invention, the bispecific antibody is a humanized antibody. In yet another embodiment of the invention, the bispecific antibody is a chimeric antibody. In an embodiment of the invention, the bispecific antibody is a combination of human, humanized and chimeric. For example, a first half antibody derived from a first parent antibody can be a human antibody and a second half antibody derived from a second parent antibody can be a humanized antibody, such that the bispecific antibody is a combination of human and humanized.

In a preferred embodiment of the invention, the bispecific antibody binds to both human and cynomolgus monkey CD37 having the sequences set forth in SEQ ID NOs: 62 and 63, respectively. This is advantageous as it allows preclinical toxicology studies to be performed in cynomolgus monkeys with the same bispecific molecule, which can then be tested in humans. If an antibody against a human target does not bind to the target in an animal model, it is very difficult to perform preclinical toxicology studies and nonclinical safety profiles of the molecule as required by regulatory authorities.

Bispecific antibody formats The present invention provides pharmaceutical compositions comprising bispecific CD37xCD37 antibodies that efficiently promote CDC and/or ADCC-mediated killing of CD37-expressing tumor cells, such as tumors of B-cell origin. Depending on the functional properties desired for a particular use, a particular antigen-binding region can be selected from the set of antibodies or antigen-binding regions described herein. Many different formats and uses of bispecific antibodies are known in the art and are discussed by Kontermann; Drug Discov Today, 2015 Jul;20(7):838-47 and MAbs, 2012 Mar-Apr;4(2):182-97.

The bispecific antibodies related to the present invention are not limited to any particular bispecific format or method of production thereof, but the bispecific antibodies should have an intact Fc domain to induce enhanced Fc-Fc interactions.

Examples of bispecific antibody molecules that can be used in the present invention include: (i) a single antibody having two arms containing different antigen-binding regions; (ii) dual variable domain antibodies (DVD-Ig), in which each light and heavy chain contains two variable domains in tandem arranged via a short peptide bond (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig ^™ ) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010)); and (iii) so-called "dock and lock" molecules based on the "dimerization and docking domain" in protein kinase A.

In one embodiment, the bispecific antibody is a crossbody, i.e. a bispecific antibody obtained via controlled Fab arm exchange (e.g. as described in WO2011131746 (Genmab)).

Examples of different classes of bispecific antibodies include, but are not limited to, (i) IgG-like molecules with complementary CH3 domains to force heterodimerization; (ii) recombinant IgG-like dual targeting molecules, in which each of the two sides of the molecule contains a Fab fragment or a portion of a Fab fragment of at least two different antibodies; (iii) IgG fusion molecules, in which a full-length IgG antibody is fused to an extra Fab fragment or a portion of a Fab fragment; (iv) Fc fusion molecules, in which a single chain Fv molecule or a stabilized diabody is fused to a heavy chain constant domain, Fc region or a portion thereof; (v) Fab fusion molecules, in which different Fab fragments are fused to each other and to a heavy chain constant domain, Fc region or a portion thereof; and (vi) scFv and diabody-based heavy chain antibodies (e.g., domain antibodies, nanobodies, etc.) in which different single chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to the Fc-.

Examples of IgG-like molecules with complementary CH3 domains include Triomab/Quadroma molecules (Trion Pharma/Fresenius Biotech; Roche, WO2011069104), so-called Knobs-into-Holes molecules (Genentech, WO9850431), CrossMAb (Roche, WO2011117329) and electrostatic steering molecules (Amgen, EP1870459 and WO2009089004; Chugai, US201000155133; Oncomed, WO2010129304), LUZ-Y molecules (Genentech, Wranik et al. J. Biol. Chem. 2012, 287(52): 43331-9, doi:10.1074/jbc.M112.397869. Epub 2012 Nov 1), DIG-body and PIG-body molecules (Pharmabcine, WO2010134666, WO2014081202), Strand Exchange Engineered Domain body (SEEDbody) molecules (EMD Serono, WO2007110205), Biclonics molecules (Merus, WO2013157953), FcΔAdp molecules (Regeneron, WO201015792), hinge-engineered bispecific IgG1 and IgG2 molecules (Pfizer/Rinat, WO11143545), Azymetric scaffold molecules (Zymeworks/Merck, WO2012058768), mAb-Fv molecules (Xencor, WO2011028952), bivalent bispecific antibodies (WO2009080254) and DuoBody® molecules (Genmab A/S, WO2011131746).

Examples of recombinant IgG-like dual targeting molecules include dual targeting (DT)-Ig molecules (WO2009058383), Two-in-one Antibodies (Genentech; Bostrom, et al 2009. Science 323, 1610-1614), Cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star, WO2008003116), Zybody molecules (Zyngenia; LaFleur et al. MAbs. 2013 Mar-Apr;5(2):208-18), common light chain approaches (Crucell/Merus, US7,262,028), κλBodies (NovImmune, WO2012023053) and CovX-body (CovX/Pfizer; Doppalapudi, V.R., et al 2007. Bioorg. Med. Chem. Lett. 17,501-506), but are not limited to these.

Examples of IgG fusion molecules include Dual Variable Domain (DVD)-Ig molecules (Abbott, US7,612,181), Dual Domain Double Headed Antibodies (Unilever; Sanofi Aventis, WO2010/0226923), IgG-like Bispecific molecules (ImClone/Eli Lilly, Lewis et al. Nat Biotechnol. 2014 Feb;32(2):191-8), Ts2Ab (MedImmune/AZ; Dimasi et al. J Mol Biol. 2009 Oct 30;393(3):672-92) and BsAb molecules (Zymogenetics, WO2010111625), HERCULES molecules (Biogen Idec, US007951918), scFv fusion molecules (Novartis), scFv fusion molecules (Changzhou Adam Biotech Inc, CN102250246) and TvAb molecules (Roche, WO2012025525, WO2012025530).

Examples of Fc fusion molecules include, but are not limited to, ScFv/Fc Fusions (Pearce et al., Biochem Mol Biol Int. 1997 Sep;42(6):1179-88), SCORPION molecules (Emergent BioSolutions/Trubion, Blankenship JW, et al. AACR 100th Annual meeting 2009 (Abstract #5465); Zymogenetics/BMS, WO2010111625), Dual Affinity Retargeting Technology (Fc-DART) molecules (MacroGenics, WO2008157379, WO2010080538) and Dual(ScFv)2-Fab molecules (National Research Center for Antibody Medicine‐China).

Examples of Fab fusion bispecific antibodies include, but are not limited to, F(ab)2 molecules (Medarex/AMGEN; Deo et al J Immunol. 1998 Feb 15;160(4):1677-86), Dual-Action or Bis-Fab molecules (Genentech, Bostrom, et al 2009. Science 323, 1610-1614), Dock-and-Lock (DNL) molecules (ImmunoMedics, WO2003074569, WO2005004809), Bivalent Bispecific molecules (Biotecnol, Schoonjans, J Immunol. 2000 Dec 15;165(12):7050-7) and Fab-Fv molecules (UCB-Celltech, WO2009040562A1).

Examples of scFv-based antibodies, diabody-based antibodies, and domain antibodies include, but are not limited to, Dual Affinity Retargeting Technology (DART) molecules (MacroGenics, WO2008157379, WO2010080538), COMBODY molecules (Epigen Biotech, Zhu et al. Immunol Cell Biol. 2010 Aug;88(6):667-75), and dual targeting nanobodies (Ablynx, Hmila et al., FASEB J. 2010).

In one aspect, the bispecific antibody comprised in the pharmaceutical composition of the present invention comprises a first Fc region comprising a first CH3 region and a second Fc region comprising a second CH3 region, wherein the sequences of the first and second CH3 regions are distinct such that the heterodimeric interaction between said first and second CH3 regions is stronger than the homodimeric interaction of said first and second CH3 regions, respectively. Further details regarding these interactions and how they may be achieved are provided in WO2011131746 and WO2013060867 (Genmab), which are incorporated herein by reference.

As further described herein, a particular method based on one homodimeric starting CD37 antibody and another homodimeric starting CD37 antibody containing a small number of fairly conservative asymmetric mutations in the CH3 region can be used to obtain stable bispecific CD37xCD37 antibodies in high yields. Asymmetric mutations mean that the sequences of the first and second CH3 regions contain amino acid substitutions at non-identical positions, such that the first and second CH3 regions have different amino acid sequences.

In one aspect, the bispecific antibody comprises a first and a second Fc region, each of the first and second Fc regions comprising at least a hinge region, a CH2 region and a CH3 region, wherein at least one amino acid in the first Fc region is substituted at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407 and K409 in a human IgG1 heavy chain, and wherein at least one amino acid in the second Fc region is substituted at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407 and K409 in a human IgG1 heavy chain, and wherein the first and second Fc regions are not substituted at the same position.

Thus, in a preferred embodiment of the invention, the first Fc region of the bispecific antibody comprises a mutation at an amino acid corresponding to position F405 in human IgG1, and the second Fc region of the bispecific antibody comprises a further mutation at an amino acid corresponding to position K409 in human IgG1. These mutations are therefore asymmetric compared to the Fc-Fc interaction enhancing mutations described above.

In one embodiment, the first Fc region has an amino acid substitution at position 366 and the second Fc region has an amino acid substitution at a position selected from the group consisting of 368, 370, 399, 405, 407, and 409. In one embodiment, the amino acid at position 366 is selected from Ala, Asp, Glu, His, Asn, Val, or Gln.

In one embodiment, the first Fc region has an amino acid substitution at position 368 and the second Fc region has an amino acid substitution at a position selected from the group consisting of 366, 370, 399, 405, 407, and 409.

In one embodiment, the first Fc region has an amino acid substitution at position 370 and the second Fc region has an amino acid substitution at a position selected from the group consisting of 366, 368, 399, 405, 407, and 409.

In one embodiment, the first Fc region has an amino acid substitution at position 399 and the second Fc region has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 405, 407, and 409.

In one embodiment, the first Fc region has an amino acid substitution at position 405 and the second Fc region has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 407, and 409.

In one embodiment, the first Fc region has an amino acid substitution at position 407 and the second Fc region has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405 and 409.

In one embodiment, the first Fc region has an amino acid substitution at position 409 and the second Fc region has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405 and 407.

Thus, in one embodiment, the sequences of the first and second Fc regions contain asymmetric mutations, i.e., mutations at different positions in the two Fc regions, e.g., a mutation at position 405 in one Fc region and a mutation at position 409 in the other Fc region.

In one embodiment, the first Fc region has an amino acid other than Lys, Leu or Met, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, at position 409, and the second Fc region has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405 and 407. In one such embodiment, the first Fc region has an amino acid other than Lys, Leu or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, and the second Fc region has an amino acid other than Phe at position 405, e.g., Gly, Ala, Val, Ile, Ser, Thr, Lys, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, Cys, Lys or Leu. In a further embodiment thereof, the first Fc region has an amino acid other than Lys, Leu or Met, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, at position 409, and the second Fc region has an amino acid other than Phe, Arg or Gly, e.g., Leu, Ala, Val, Ile, Ser, Thr, Met, Lys, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, at position 405.

In another embodiment, the first Fc region comprises Phe at position 405 and an amino acid other than Lys, Leu or Met, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, at position 409, and the second Fc region comprises an amino acid other than Phe, e.g., Gly, Ala, Val, Ile, Ser, Thr, Lys, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, Leu, Met or Cys, at position 405 and Lys at position 409. In a further embodiment thereof, the first Fc region comprises Phe at position 405 and an amino acid other than Lys, Leu or Met, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, at position 409, and the second Fc region comprises an amino acid other than Phe, Arg or Gly, e.g., Leu, Ala, Val, Ile, Ser, Thr, Met, Lys, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, at position 405 and Lys at position 409.

In another embodiment, the first Fc region comprises Phe at position 405 and an amino acid other than Lys, Leu or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, and the second Fc region comprises Leu at position 405 and Lys at position 409. In a further embodiment thereof, the first Fc region has Phe at position 405 and includes Args at position 409, and the second Fc region includes an amino acid other than Phe, Arg, or Gly, e.g., Leu, Ala, Val, Ile, Ser, Thr, Lys, Met, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys at position 405 and includes Lys at position 409. In another embodiment, the first Fc region includes Phe at position 405 and Arg at position 409, and the second Fc region includes Leu at position 405 and Lys at position 409.

In a further embodiment, the first Fc region comprises an amino acid other than Lys, Leu or Met, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, at position 409, and the second Fc region comprises Lys at position 409, Thr at position 370, and Leu at position 405. In a further embodiment, the first Fc region comprises Arg at position 409, and the second Fc region comprises Lys at position 409, Thr at position 370, and Leu at position 405.

In yet a further embodiment, the first Fc region comprises Lys at position 370, Phe at position 405, and Arg at position 409, and the second Fc region comprises Lys at position 409, Thr at position 370, and Leu at position 405.

In another embodiment, the first Fc region comprises an amino acid other than Lys, Leu or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, and the second Fc region comprises Lys at position 409, a) Ile at position 350 and Leu at position 405, or b) Thr at position 370 and Leu at position 405.

In another embodiment, the first Fc region comprises Arg at position 409 and the second Fc region comprises Lys at position 409 and a) Ile at position 350 and Leu at position 405, or b) Thr at position 370 and Leu at position 405.

In another embodiment, the first Fc region comprises Thr at position 350, Lys at position 370, Phe at position 405, and Arg at position 409, and the second Fc region comprises Lys at position 409, a) Ile at position 350 and Leu at position 405, or b) Thr at position 370 and Leu at position 405.

In another embodiment, the first Fc region comprises Thr at position 350, Lys at position 370, Phe at position 405, and Arg at position 409, and the second Fc region comprises Ile at position 350, Thr at position 370, Leu at position 405, and Lys at position 409.

In one embodiment, the first Fc region has an amino acid other than Lys, Leu or Met at position 409 and the second Fc region has an amino acid other than Phe, e.g., other than Phe, Arg or Gly, at position 405; or the first CH3 region has an amino acid other than Lys, Leu or Met at position 409 and the second CH3 region has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at position 407.

In one embodiment, the bispecific antibody comprises a first Fc region having an amino acid at position 409 other than Lys, Leu or Met, and a second Fc region having an amino acid at position 407 other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr.

In one embodiment, the bispecific antibody comprises a first Fc region having Tyr at position 407 and an amino acid other than Lys, Leu or Met at position 409, and a second Fc region having an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at position 407 and Lys at position 409.

In one embodiment, the bispecific antibody comprises a first Fc region having Tyr at position 407 and Arg at position 409, and a second Fc region having an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser, or Thr at position 407 and Lys at position 409.

In another embodiment, the first Fc region has an amino acid other than Lys, Leu or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, and the second Fc region has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at position 407, e.g., Leu, Met, Gly, Ala, Val, Ile, His, Asn, Pro, Trp or Cys. In another embodiment, the first Fc region has an amino acid other than Lys, Leu or Met, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, at position 409, and the second Fc region has Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407.

In another embodiment, the first Fc region has an amino acid other than Lys, Leu or Met, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, at position 409, and the second Fc region has Gly, Leu, Met, Asn or Trp at position 407.

In another embodiment, the first Fc region has Tyr at position 407 and an amino acid other than Lys, Leu or Met, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, at position 409, and the second Fc region has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr, e.g., Leu, Met, Gly, Ala, Val, Ile, His, Asn, Pro, Trp or Cys, at position 407 and Lys at position 409.

In another embodiment, the first Fc region has Tyr at 407 and an amino acid other than Lys, Leu or Met, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, at position 409, and the second Fc region has Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407 and Lys at 409.

In another embodiment, the first Fc region has Tyr at position 407 and an amino acid other than Lys, Leu or Met, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, at position 409, and the second Fc region has Gly, Leu, Met, Asn or Trp at position 407 and Lys at position 409.

In another embodiment, the first Fc region has Tyr at position 407 and Arg at position 409, and the second Fc region has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr, e.g., Leu, Met, Gly, Ala, Val, Ile, His, Asn, Pro, Trp or Cys, at position 407 and Lys at position 409.

In another embodiment, the first Fc region has Tyr at position 407 and Arg at position 409, and the second Fc region has Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407 and Lys at position 409.

In another embodiment, the first Fc region has Tyr at position 407 and Arg at position 409, and the second Fc region has Gly, Leu, Met, Asn or Trp at position 407 and Lys at position 409.

In another embodiment, the first Fc region has an amino acid other than Lys, Leu or Met, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, at position 409, and the second Fc region has
(i) having an amino acid other than Phe, Leu and Met at position 368, such as Gly, Ala, Val, Ile, Ser, Thr, Lys, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, or (ii) having Trp at position 370, or (iii) having an amino acid other than Asp, Cys, Pro, Glu or Gln at position 399, such as Phe, Leu, Met, Gly, Ala, Val, Ile, Ser, Thr, Lys, Arg, His, Asn, Trp, Tyr or Cys, or (iv) having an amino acid other than Lys, Arg, Ser, Thr or Trp at position 366, e.g., Phe, Leu, Met, Ala, Val, Gly, Ile, Asn, His, Asp, Glu, Gln, Pro, Tyr or Cys.

In one embodiment, the first Fc region has an Arg, Ala, His or Gly at position 409 and the second Fc region has
(i) having Lys, Gln, Ala, Asp, Glu, Gly, His, Ile, Asn, Arg, Ser, Thr, Val or Trp at position 368, or (ii) having Trp at position 370, or (iii) having Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, Trp, Phe, His, Lys, Arg or Tyr at position 399, or (iv) having Ala, Asp, Glu, His, Asn, Val, Gln, Phe, Gly, Ile, Leu, Met or Tyr at position 366.

In one embodiment, the first Fc region has an Arg at position 409 and the second Fc region has
(i) having Asp, Glu, Gly, Asn, Arg, Ser, Thr, Val or Trp at position 368, or (ii) having Trp at position 370, or (iii) having Phe, His, Lys, Arg or Tyr at position 399, or (iv) having Ala, Asp, Glu, His, Asn, Val, Gln at position 366.

In addition to the amino acid substitutions specified above, the first and second Fc regions may contain further amino acid substitutions, deletions or insertions relative to the wild-type Fc sequence.

In a preferred embodiment of the present invention, the second Fc region of the bispecific antibody contains a mutation corresponding to F405 in human IgG1 when using EU numbering, and the first Fc region contains a mutation corresponding to K409 in human IgG1.

In one embodiment, the mutations at positions F405 and K409 are substitutions. In a particular embodiment, the substitution at position F405 is a F405L substitution. In another embodiment, the substitution at position K409 is a K409R substitution.

In a preferred embodiment,
a) the first Fc region comprises an additional mutation corresponding to F405L when using EU numbering in human IgG1, and the second Fc region comprises an additional mutation corresponding to K409R when using EU numbering in human IgG1, or
b) the second Fc region comprises an additional mutation corresponding to F405L when using EU numbering in human IgG1, and the first Fc region comprises an additional mutation corresponding to K409R when using EU numbering in human IgG1.

In embodiments in which the bispecific antibody is an IgG4 isotype, the first Fc region may further comprise an F405L substitution and an R409K substitution. In such embodiments, the second Fc region is not substituted at either the 405 or 409 amino acid positions.

Unless expressly stated otherwise, it will be understood that all of the amino acid mutations at the disclosed positions are mutations relative to human IgG1, using human IgG1 for numbering using the EU numbering system.

In one embodiment of the invention, the first or second Fc region comprises a sequence selected from the group consisting of SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, and SEQ ID NO: 135. In one embodiment of the invention, the first and second Fc regions comprise a sequence selected from the group consisting of SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, and SEQ ID NO: 135.

In one embodiment of the invention, the first Fc region comprises the sequence set forth in SEQ ID NO: 128 and the second Fc region comprises the sequence set forth in SEQ ID NO: 129, or vice versa. In one embodiment of the invention, the first Fc region comprises the sequence set forth in SEQ ID NO: 130 and the second Fc region comprises the sequence set forth in SEQ ID NO: 133, or vice versa. In one embodiment of the invention, the first Fc region comprises the sequence set forth in SEQ ID NO: 131 and the second Fc region comprises the sequence set forth in SEQ ID NO: 134, or vice versa. In one embodiment of the invention, the first Fc region comprises the sequence set forth in SEQ ID NO: 132 and the second Fc region comprises the sequence set forth in SEQ ID NO: 135, or vice versa.

In one embodiment, neither the first nor the second Fc region contains a Cys-Pro-Ser-Cys sequence in the core hinge region.

In a further embodiment, both the first and second Fc regions contain a Cys-Pro-Pro-Cys sequence in the core hinge region.

The present specification provides a bispecific antibody that can be produced with high yield and is stable in vivo.

In another embodiment, the bispecific antibody has enhanced CDC and/or ADCC effector function compared to the same bispecific antibody without the Fc-Fc interaction enhancing mutations. In another embodiment, the bispecific antibody used in the present invention has enhanced CDC and/or ADCC effector function compared to a parent monoclonal antibody having either the first or second binding domain of the bispecific antibody and having the same Fc-Fc interaction enhancing mutations as the bispecific antibody used in the present invention.

In one embodiment of the pharmaceutical composition of the present invention, the bispecific antibody comprises a heavy chain as set forth in SEQ ID NOs: 118 and 120 and a light chain as set forth in SEQ ID NOs: 119 and 121, wherein the heavy chain as set forth in SEQ ID NO: 118 forms an antigen-binding region together with the light chain as set forth in SEQ ID NO: 119, and the heavy chain as set forth in SEQ ID NO: 120 forms an antigen-binding region together with the light chain as set forth in SEQ ID NO: 121.

In a preferred embodiment of the pharmaceutical composition of the present invention, the bispecific antibody comprises a heavy chain as set forth in SEQ ID NOs: 124 and 125 and a light chain as set forth in SEQ ID NOs: 119 and 126, wherein the heavy chain as set forth in SEQ ID NO: 124 forms an antigen-binding region together with the light chain as set forth in SEQ ID NO: 119, and the heavy chain as set forth in SEQ ID NO: 125 forms an antigen-binding region together with the light chain as set forth in SEQ ID NO: 126.

Methods for Preparing Bispecific Antibodies Conventional methods such as hybrid hybridoma and chemical conjugation methods (Marvin and Zhu (2005) Acta Pharmacol Sin 26:649) can be used to prepare the bispecific antibodies contained in the pharmaceutical compositions of the invention. Co-expression in a host cell of two antibodies consisting of different heavy and light chains gives rise to a mixture of possible antibody products in addition to the desired bispecific antibody, which can be isolated, for example, by affinity chromatography or similar methods.

Strategies that favor the formation of functional bispecific products by co-expression of different antibody constructs can also be used, such as the method described by Lindhofer et al. (1995 J Immunol 155:219). Fusion of rat and mouse hybridomas producing different antibodies results in only a limited number of heterodimeric proteins due to preferential species-restricted heavy/light chain pairing. Another strategy to promote heterodimer formation over homodimers is the "knob-into-hole" strategy, in which a protrusion is introduced into a first heavy chain polypeptide and a corresponding cavity is introduced into a second heavy chain polypeptide, such that the protrusion is positioned into the cavity at the interface of these two heavy chains, promoting heterodimer formation and preventing homodimer formation. The "protrusion" is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain. A complementary "cavity" of the same or similar size as the protrusion is created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (US Pat. No. 5,731,168). EP1870459 (Chugai) and WO2009089004 (Amgen) describe other strategies to favor heterodimer formation by co-expression of different antibody domains in a host cell. In these methods, one or more residues constituting the CH3-CH3 interface in both CH3 domains are replaced with charged amino acids such that homodimer formation is electrostatically unfavorable and heterodimerization is electrostatically favored. WO2007110205 (Merck) describes yet another strategy that exploits the differences in the CH3 domains of IgA and IgG to promote heterodimerization.

Another in vitro method for making bispecific antibodies is described in WO2008119353 (Genmab), in which bispecific antibodies are formed by "Fab arm" or "half molecule" exchange (exchange of heavy chains and associated light chains) between two monospecific IgG4 or IgG4-like antibodies by incubation under reducing conditions. The resulting product is a bispecific antibody with two Fab arms that may contain different sequences.

A preferred method for preparing a bispecific CD37xCD37 antibody is the method described in WO2011131746 and WO2013060867 (Genmab), comprising the following steps:
a) providing a first antibody comprising an Fc region that comprises a first CH3 region;
b) providing a second antibody comprising a second Fc region comprising a second CH3 region, wherein the first antibody is a CD37 antibody and the second antibody is a different CD37 antibody; and wherein the sequences of the first and second CH3 regions are different such that a heterodimeric interaction between the first and second CH3 regions is stronger than a homodimeric interaction of each of the first and second CH3 regions;
c) incubating the first antibody with the second antibody under reducing conditions; and
d) obtaining the bispecific antibody.

In one embodiment, the first antibody and the second antibody are incubated under reducing conditions sufficient to allow cysteines in the hinge region to undergo disulfide bond isomerization, and the heterodimeric interaction between the first and second antibodies in the resulting heterodimeric antibody is such that no Fab arm exchange occurs after 24 hours at 37°C with 0.5 mM GSH.

Without being bound by theory, in step c) the heavy chain disulfide bonds in the hinge region of the parent antibody are reduced and the resulting cysteines can form inter-heavy chain disulfide bonds with cysteine residues of another parent antibody molecule (originally with a different specificity). In one embodiment of the method, the reducing conditions in step c) comprise the addition of a reducing agent, e.g. a reducing agent selected from the group consisting of 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine and beta-mercaptoethanol, preferably a reducing agent selected from the group consisting of 2-mercaptoethylamine, dithiothreitol and tris(2-carboxyethyl)phosphine. In a further embodiment, step c) comprises a step of returning the conditions to non-reducing or less reducing, e.g. by removal of the reducing agent, e.g. by desalting.

In this method, any of the CD37 antibodies described herein may be used, including first and second CD37 antibodies comprising a first and/or second Fc region. Examples of such first and second Fc regions may include any of those described herein, including combinations of such first and second Fc regions.

In one embodiment of this method, the first and/or second antibody is a full-length antibody.

The Fc regions of the first and second antibodies can be of any isotype, including IgG1, IgG2, IgG3, or IgG4. In one embodiment of this method, the Fc regions of both the first and second antibodies are of IgG1 isotype. In another embodiment, one of the antibody Fc regions is of IgG1 isotype and the other is of IgG4 isotype. In the latter embodiment, the resulting bispecific antibody contains an IgG1 Fc region and an IgG4 Fc region and may therefore have interesting intermediate properties with respect to activation of effector functions.

In a further embodiment, one of the starting antibody proteins is engineered not to bind Protein A, thus allowing separation of the heterodimeric protein from the homodimeric starting protein by passing the product through a Protein A column.

As noted above, the sequences of the first and second CH3 regions of the homodimeric starting antibody (parent antibody) are different such that the heterodimeric interaction between the first and second CH3 regions is stronger than the homodimeric interaction of the first and second CH3 regions, respectively. Further details regarding these interactions and how they can be achieved are provided in WO2011131746 and WO2013060867 (Genmab), which are incorporated herein by reference in their entirety.

In particular, stable bispecific CD37xCD37 antibodies can be obtained in high yields using the above method based on two homodimeric starting antibodies that bind to different epitopes of CD37 and contain a small number of fairly conservative asymmetric mutations in the CH3 regions. By asymmetric mutations, it is meant that the sequences of the first and second CH3 regions contain amino acid substitutions at non-identical positions.

Bispecific antibodies may also be obtained by co-expression in a single cell of constructs encoding the first and second polypeptides. Such a method may comprise the following steps:
a) providing a first nucleic acid construct encoding a first polypeptide comprising a first Fc region (including a first CH3 region) and a first antigen-binding region of a first antibody heavy chain;
b) providing a second nucleic acid construct encoding a second polypeptide comprising a second Fc region (including a second CH3 region) and a second antigen-binding region of a second antibody heavy chain, wherein the sequences of said first and second CH3 regions are different such that a heterodimeric interaction between said first and second CH3 regions is stronger than a homodimeric interaction of said first and second CH3 regions, respectively, and optionally said first and second nucleic acid constructs encoding light chain sequences of said first and second antibodies;
c) co-expressing the first and second nucleic acid constructs in a host cell; and
d) Obtaining said heterodimeric protein from the cell culture.

In one embodiment, a bispecific antibody as defined in any of the embodiments disclosed herein comprises a first Fc region and a second Fc region, wherein neither the first nor the second Fc region comprises a Cys-Pro-Ser-Cys sequence in the hinge region.

In one embodiment, a bispecific antibody as defined in any of the embodiments disclosed herein comprises a first Fc region and a second Fc region, both of which comprise a Cys-Pro-Pro-Cys sequence in the hinge region.

In one embodiment, a bispecific antibody as defined in any of the embodiments disclosed herein comprises a first Fc region and a second Fc region, wherein the first and second Fc regions are human antibody Fc regions.

In one embodiment, a bispecific antibody as defined in any of the embodiments disclosed herein comprises a first Fc region and a second Fc region, and the first and second antigen-binding regions comprise a human antibody VH sequence and optionally a human antibody VL sequence.

In one embodiment, a bispecific antibody as defined in any of the embodiments disclosed herein comprises a first Fc region and a second Fc region, and the first and second antigen-binding regions comprise first and second light chains.

Expression vectors (including promoters, enhancers, etc.) and host cells suitable for producing antibodies are well known in the art. Examples of host cells include yeast cells, bacterial cells, and mammalian cells (e.g., CHO cells or HEK cells).

Thus, an expression vector in the context of the present invention can be any suitable vector, including chromosomal, non-chromosomal and synthetic nucleic acid vectors (nucleic acid sequences that include an appropriate set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the nucleic acid encoding the CD37 antibody is comprised within, for example, a naked DNA or RNA vector comprising a linear expression element (e.g., as described in Sykes and Johnston, Nat Biotech 17, 355 59 (1997)), a compacted nucleic acid vector (e.g., as described in US 6,077,835 and/or WO 00/70087), a plasmid vector, e.g., pBR322, pUC19/18 or pUC118/119, a "midge" minimum size nucleic acid vector (e.g., as described in Schakowski et al., Mol Ther 3, 793 800 (2001)), or a precipitated nucleic acid vector construct, e.g., a CaPO4 precipitated construct (e.g., as described in WO200046147, Benvenisty and Reshef, PNAS USA 83, 9551 55 (1986), Wigler et al., Cell 14, 725 (1978) and Coraro and Pearson, Somatic Cell Genetics 7, 603 (1981). Such nucleic acid vectors and their uses are well known in the art (see, e.g., US 5,589,466 and US 5,973,972).

The vector may be suitable for expression of the antibody in bacterial cells. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J Biol Chem 264, 5503 5509 (1989)), pET vectors (Novagen, Madison WI), etc.

Also or alternatively, the expression vector may be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be used. Suitable vectors include, for example, vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH (reviewed in F. Ausubel et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987) and Grant et al., Methods in Enzymol 153, 516 544 (1987)).

Also or alternatively, the expression vector may be a vector suitable for expression in mammalian cells, e.g., a vector containing glutamine synthetase as a selection marker, e.g., a vector described in Bebbington (1992) Biotechnology (NY) 10:169-175.

The nucleic acid and/or vector may also include a nucleic acid sequence encoding a secretion/localization sequence capable of directing a polypeptide (e.g., a nascent polypeptide chain) into the periplasmic space or into the cell medium. Such sequences are known in the art and include secretory leaders or signal peptides.

The expression vector may contain or be associated with any suitable promoter, enhancer and other expression promoting elements. Examples of such elements include strong expression promoters (e.g., human CMV IE promoter/enhancer and RSV, SV40, SL33, MMTV and HIV LTR promoters), efficient poly(A) termination sequences, an origin of replication for the plasmid product in E. coli, antibiotic resistance genes as selectable markers and/or convenient cloning sites (e.g., polylinkers). The nucleic acid may also contain an inducible promoter as opposed to a constitutive promoter such as CMV IE.

In one embodiment, an expression vector encoding a CD37 antibody can be placed and/or delivered into a host cell or animal via a viral vector.

Further aspects of the invention In another aspect, the invention relates to a composition comprising a bispecific antibody of the invention and further comprising a monospecific anti-CD37 antibody, preferably an anti-CD37 antibody having an antigen-binding region of either the first or the second antigen-binding region of the bispecific antibody.

In another aspect, the present invention relates to a composition of the present invention for use as a medicament.

In one aspect, the present invention relates to a pharmaceutical composition of the present invention for use in treating cancer, an autoimmune disease, or an inflammatory disorder.

In another aspect, the present invention relates to a pharmaceutical composition of the present invention for use in the treatment of allergy, graft rejection, or B-cell malignancies, such as non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), mantle cell lymphoma (MCL), plasma cell leukemia (PCL), diffuse large B-cell lymphoma (DLBCL) or acute lymphoblastic leukemia (ALL).

In one embodiment, the pharmaceutical compositions for use according to the invention are administered parenterally, e.g., subcutaneously, intramuscularly, or intravenously.

In another aspect, the present invention relates to a method for treating rheumatoid arthritis, such as acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immune-mediated arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, spondyloarthritis and juvenile rheumatoid arthritis, osteoarthritis, progressive chronic arthritis, osteoarthritis, primary chronic polyarthritis, reactive arthritis and ankylosing spondylitis, systemic lupus erythematosus (SLE), such as cutaneous SLE or relates to the pharmaceutical composition of the present invention for use in the treatment of subacute cutaneous SLE, neonatal lupus syndrome (NLE) and disseminated lupus erythematosus, multiple sclerosis, inflammatory bowel diseases (IBD) including ulcerative colitis and Crohn's disease, chronic obstructive pulmonary disease (COPD), psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis, diabetes mellitus, Raynaud's syndrome and glomerulonephritis, palmoplantar pustulosis (PPP), erosive lichen planus, bullous pemphigus, epidermolysis bullosa, contact dermatitis and atopic dermatitis, polyneuritis including Guillain-Barre syndrome.

In another aspect, the present invention relates to a pharmaceutical composition of the present invention for use in treating allergy, transplant rejection, or B-cell malignancies.

In another aspect, the present invention relates to a pharmaceutical composition of the present invention for use in combination with one or more additional therapeutic agents. The one or more additional therapeutic agents may be selected from the group including, for example, doxorubicin, cisplatin, bleomycin, carmustine, cyclophosphamide, chlorambucil, bendamustine, vincristine, fludarabine, ibrutinib, and anti-CD20 antibodies (e.g., rituximab, ofatumumab, obinutuzumab, veltuzumab, ocaratuzumab, ocrelizumab, or TRU-015).

In a preferred embodiment, the additional therapeutic agent is an anti-CD20 antibody. In one embodiment, the anti-CD20 antibody is capable of binding to human CD20 having the sequence set forth in SEQ ID NO: 72. In one embodiment, the anti-CD20 antibody is capable of binding to cynomolgus CD20 having the sequence set forth in SEQ ID NO: 73. In one embodiment, the anti-CD20 antibody is capable of binding to human and cynomolgus CD20 having the sequences set forth in SEQ ID NOs: 72 and 73, respectively.

In one embodiment, the anti-CD20 antibody is capable of binding to an epitope on human CD20 that does not include or require amino acid residues alanine at position 170 or proline at position 172, but does include or require amino acid residues asparagine at position 163 and asparagine at position 166 of SEQ ID NO: 72. Examples of such antibodies are the antibodies designated 2F2 and 7D8, disclosed in WO2004035607 (Genmab), and the antibody designated 2C6, disclosed in WO2005103081 (Genmab). The CDR sequences of 7D8 are disclosed in Table 1.

In one embodiment, the anti-CD20 antibody can bind to an epitope on human CD20 that does not include or require amino acid residues alanine at position 170 or proline at position 172 of SEQ ID NO: 72. An example of such an antibody is 11B8, disclosed in WO2004035607 (Genmab). The CDR sequences of 11B8 are disclosed in Table 1.

In one embodiment, the anti-CD20 antibody can bind to a discontinuous epitope on human CD20, the epitope comprising a portion of the first small extracellular loop and a portion of the second extracellular loop.

In one embodiment, the anti-CD20 antibody can bind to a discontinuous epitope on human CD20, the epitope having residues AGIYAP in the first small extracellular loop and residues MESLNFIRAHTPY in the second extracellular loop.

Anti-CD20 antibodies can be characterized as type I and type II anti-CD20 antibodies. Type I anti-CD20 antibodies have high CDC and ADCC activity but low apoptotic activity (e.g., ofatumumab (2F2) and rituximab), whereas type II CD20 antibodies have low or no CDC activity but high ADCC and apoptotic activity (e.g., obinutuzumab and 11B8). Type I antibodies also induce CD20 to redistribute into large detergent-resistant microdomains (rafts), whereas type II antibodies do not.

In one embodiment, the anti-CD20 antibody comprises an antigen-binding region capable of binding to human CD20, and the antigen-binding region competes for binding to human CD20 with an anti-CD20 antibody comprising a variable heavy chain (VH) sequence and a variable light chain (VL) sequence set forth in SEQ ID NO: 74 and SEQ ID NO: 78, respectively.

In one embodiment, the anti-CD20 antibody comprises an antigen-binding region capable of binding to human CD20, and the antigen-binding region competes for binding to human CD20 with an anti-CD20 antibody comprising a variable heavy chain (VH) sequence and a variable light chain (VL) sequence set forth in SEQ ID NO: 81 and SEQ ID NO: 109, respectively.

In one embodiment, the anti-CD20 antibody comprises an antigen-binding region capable of binding to human CD20, and the antigen-binding region competes for binding to human CD20 with an anti-CD20 antibody comprising a variable heavy chain (VH) sequence and a variable light chain (VL) sequence set forth in SEQ ID NO: 94 and SEQ ID NO: 98, respectively.

In one embodiment, the anti-CD20 antibody comprises an antigen-binding region capable of binding to human CD20, and the antigen-binding region competes for binding to human CD20 with an anti-CD20 antibody comprising a variable heavy chain (VH) sequence and a variable light chain (VL) sequence set forth in SEQ ID NO: 87 and SEQ ID NO: 91, respectively.

In one embodiment, the anti-CD20 antibody comprises an antigen-binding region capable of binding to human CD20, and the antigen-binding region competes for binding to human CD20 with an anti-CD20 antibody comprising a variable heavy chain (VH) sequence and a variable light chain (VL) sequence set forth in SEQ ID NO: 101 and SEQ ID NO: 105, respectively.

In one embodiment, the anti-CD20 antibody has the CDR sequence:
VH CDR1 sequence as set forth in SEQ ID NO: 75;
VH CDR2 sequence as set forth in SEQ ID NO: 76;
VH CDR3 sequence as set forth in SEQ ID NO: 77;
The VL CDR1 sequence set forth in SEQ ID NO: 79;
a VL CDR2 sequence which is a DAS, and
VL CDR3 sequence according to SEQ ID NO: 80 [7D8]
The antigen-binding region capable of binding to human CD20 comprises:

In one embodiment, the anti-CD20 antibody has the CDR sequence:
VH CDR1 sequence as set forth in SEQ ID NO: 82;
VH CDR2 sequence as set forth in SEQ ID NO: 83;
VH CDR3 sequence as set forth in SEQ ID NO: 84;
VL CDR1 sequence as set forth in SEQ ID NO: 85;
a VL CDR2 sequence which is a DAS, and
VL CDR3 sequence according to SEQ ID NO: 86 [118B]
The antigen-binding region capable of binding to human CD20 comprises:

In one embodiment, the anti-CD20 antibody has the CDR sequence:
VH CDR1 sequence as set forth in SEQ ID NO: 95;
VH CDR2 sequence as set forth in SEQ ID NO: 96;
VH CDR3 sequence as set forth in SEQ ID NO: 97;
VL CDR1 sequence as set forth in SEQ ID NO: 99;
a VL CDR2 sequence that is an ATS, and
It comprises an antigen-binding region capable of binding to human CD20 comprising the VL CDR3 sequence set forth in SEQ ID NO: 100. [Rituximab]

In one embodiment, the anti-CD20 antibody has the CDR sequence:
VH CDR1 sequence as set forth in SEQ ID NO: 88;
VH CDR2 sequence as set forth in SEQ ID NO: 89;
VH CDR3 sequence as set forth in SEQ ID NO: 90;
VL CDR1 sequence as set forth in SEQ ID NO: 92;
a VL CDR2 sequence which is a DAS, and
Ofatumumab comprises an antigen-binding region capable of binding to human CD20 comprising the VL CDR3 sequence set forth in SEQ ID NO: 93.

In one embodiment, the anti-CD20 antibody has the CDR sequence:
VH CDR1 sequence as set forth in SEQ ID NO: 102;
VH CDR2 sequence as set forth in SEQ ID NO: 103;
VH CDR3 sequence as set forth in SEQ ID NO: 104;
The VL CDR1 sequence set forth in SEQ ID NO: 106;
a VL CDR2 sequence that is QMS, and
It comprises an antigen-binding region capable of binding to human CD20 comprising the VL CDR3 sequence set forth in SEQ ID NO: 107. [Obinutuzumab]

In one embodiment, the anti-CD20 antibody is
i) the VH CDR1 sequence set forth in SEQ ID NO: 75;
VH CDR2 sequence as set forth in SEQ ID NO: 76;
VH CDR3 sequence as set forth in SEQ ID NO: 77;
The VL CDR1 sequence set forth in SEQ ID NO: 79;
a VL CDR2 sequence which is a DAS, and
VL CDR3 sequence set forth in SEQ ID NO: 80 [7D8];
ii) the VH CDR1 sequence set forth in SEQ ID NO: 82;
VH CDR2 sequence as set forth in SEQ ID NO: 83;
VH CDR3 sequence as set forth in SEQ ID NO: 84;
VL CDR1 sequence as set forth in SEQ ID NO: 85;
a VL CDR2 sequence which is a DAS, and
VL CDR3 sequence set forth in SEQ ID NO: 86 [118B];
iii) the VH CDR1 sequence set forth in SEQ ID NO: 95;
VH CDR2 sequence as set forth in SEQ ID NO: 96;
VH CDR3 sequence as set forth in SEQ ID NO: 97;
VL CDR1 sequence as set forth in SEQ ID NO: 99;
a VL CDR2 sequence which is an ATS, and
VL CDR3 sequence set forth in SEQ ID NO: 100 [rituximab];
iv) the VH CDR1 sequence set forth in SEQ ID NO: 88;
VH CDR2 sequence as set forth in SEQ ID NO: 89;
VH CDR3 sequence as set forth in SEQ ID NO: 90;
VL CDR1 sequence as set forth in SEQ ID NO: 92;
a VL CDR2 sequence which is a DAS, and
VL CDR3 sequence set forth in SEQ ID NO: 93 [ofatumumab]; and
v) the VH CDR1 sequence set forth in SEQ ID NO: 102;
VH CDR2 sequence as set forth in SEQ ID NO: 103;
VH CDR3 sequence as set forth in SEQ ID NO: 104;
The VL CDR1 sequence set forth in SEQ ID NO: 106;
a VL CDR2 sequence that is QMS, and
VL CDR3 sequence according to SEQ ID NO: 107 [obinutuzumab]
The antibody comprises an antigen-binding region capable of binding to human CD20, the antigen-binding region comprising a CDR sequence selected from the group consisting of:

In another aspect, the present invention relates to the use of the pharmaceutical composition of the present invention for the manufacture of a medicament. In another aspect thereof, the use is directed to the treatment of cancer, autoimmune diseases, or inflammatory disorders, such as allergies, graft rejection, or B-cell malignancies, such as non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), mantle cell lymphoma (MCL), plasma cell leukemia (PCL), diffuse large B-cell lymphoma (DLBCL) or acute lymphoblastic leukemia (ALL), rheumatoid arthritis, such as acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immune arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, spondyloarthritis and juvenile rheumatoid arthritis. Use for the manufacture of a medicament for the treatment of arthritis, osteoarthritis, progressive chronic arthritis, osteoarthritis, primary chronic polyarthritis, reactive arthritis and ankylosing spondylitis, systemic lupus erythematosus (SLE), e.g. cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE) and disseminated lupus erythematosus, multiple sclerosis, inflammatory bowel disease (IBD) including ulcerative colitis and Crohn's disease, chronic obstructive pulmonary disease (COPD), psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis, diabetes, Raynaud's syndrome and glomerulonephritis, palmoplantar pustulosis (PPP), erosive lichen planus, bullous pemphigus, epidermolysis bullosa, contact dermatitis and atopic dermatitis, polyradiculitis including Guillain-Barre syndrome.

In one embodiment of these uses of the invention, the pharmaceutical composition is for parenteral administration, e.g., subcutaneous, intramuscular, or intravenous administration.

In further embodiments of these uses of the invention, the treatment comprises combination therapy with one or more further therapeutic agents, for example selected from the group including doxorubicin, cisplatin, bleomycin, carmustine, cyclophosphamide, chlorambucil, bendamustine, vincristine, fludarabine, ibrutinib, and anti-CD20 antibodies, for example rituximab or ofatumumab.

In another aspect, the present invention relates to a method for inducing cell death or inhibiting growth and/or proliferation of tumor cells expressing CD37, comprising administering to an individual in need thereof an effective amount of a pharmaceutical composition of the present invention. In a particular embodiment, the method is a method for treating an individual having allergy, graft rejection, or a B-cell malignancy, such as non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), mantle cell lymphoma (MCL), plasma cell leukemia (PCL), diffuse large B-cell lymphoma (DLBCL) or acute lymphoblastic leukemia (ALL), comprising administering to the individual an effective amount of a pharmaceutical composition of the present invention. In certain embodiments, the method includes administering one or more additional therapeutic agents, such as doxorubicin, cisplatin, bleomycin, carmustine, cyclophosphamide, chlorambucil, bendamustine, vincristine, fludarabine, ibrutinib, or an anti-CD20 antibody (e.g., rituximab, ofatumumab, obinutuzumab, veltuzumab, ocaratuzumab, ocrelizumab, or TRU-015) in combination with the antibody or bispecific antibody.

In one embodiment, the pharmaceutical composition is administered parenterally, e.g., subcutaneously, intramuscularly, or intravenously.

In one embodiment of the invention, the further therapeutic agent is selected from the group consisting of cyclophosphamide, chlorambucil, bendamustine, ifosfamide, cisplatin, carboplatin, oxaliplatin, carmustine, prednisone, dexamethasone, fludarabine, pentostatin, cladribine, fluorouracil, gemcitabine, cytarabine, methotrexate, pralatrexate, gemcitabine, vincristine, paclitaxel, docetaxel, doxorubicin, mitoxantrone, etoposide, topotecan, irinotecan, bleomycin, CD20-specific rituximab, obinutuzumab, and ofatumumab, CD52-specific alemtuzumab, and CD30-specific brentuximab. , JNJ-63709178, JNJ-64007957, HuMax-IL8, anti-DR5, anti-VEGF, anti-CD38, anti-PD-1, anti-PD-L1, anti-CTLA4, anti-CD40, anti-CD137, anti-GITR, anti-VISTA, antibodies specific for other immune-modulatory targets, brentuximab vedotin, HuMax-TAC-ADC, interferon, thalidomide, lenalidomide, axicabtagene ciloleucel, bortezomib, romidepsin, belinostat, vorinostat, ibrutinib, acalabrutinib, idelalisib, copanlisib, sorafenib, sunitinib, everolimus, recombinant human TRAIL, birinapant, and venetoclax.

In one embodiment of the invention, the further therapeutic agent is selected from the group including ibrutinib, rituximab, venetoclax, CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone), bendamustine, fludarabine, cyclophosphamide, and chlorambucil.

In one embodiment of the invention, the further therapeutic agent is selected from the group including ibrutinib, rituximab, and venetoclax.

Sequence (Table 1)

Example 1: Generation of CD37-specific antibodies in rabbits
Expression construct for CD37
The following codon-optimized constructs were generated for the expression of full-length CD37 variants: human (Homo sapiens) CD37 (Genbank accession number NP_001765) (SEQ ID NO: 62), cynomolgus monkey (Macaca fascicularis) CD37 (mfCD37) (SEQ ID NO: 63). In addition, the following codon-optimized constructs were generated for the expression of various CD37 ECD variants: a signal peptide coding sequence followed by the second extracellular domain (EC2) (aa 112-241) of human CD37 fused to the Fc (CH2-CH3) domain of human IgG with a C-terminal His tag (CD37EC2-FcHis, SEQ ID NO: 64) and a similar construct for mfCD37 (CD37mfEC2-FcHis, SEQ ID NO: 65). The constructs contained appropriate restriction sites for cloning and an optimal Kozak (GCCGCCACC) sequence [Kozak et al. (1999) Gene 234: 187-208]. The constructs were cloned into the mammalian expression vector pcDNA3.3 (Invitrogen) or an equivalent vector.

Transient expression in CHO and HEK cells Membrane proteins were transiently expressed in Freestyle 293-F (HEK293F) cells (Life technologies, USA) using 293fectin (Life technologies) essentially as described by the manufacturer, or in Freesyle CHO-S cells (CHO) (Life technologies) using Freestyle Max reagent (Life technologies) essentially as described by the manufacturer. Soluble proteins were transiently expressed in Expi293 cells (Life technologies) using ExpiFectamine 293 reagent (Life technologies) essentially as described by the manufacturer.

Fc fusion proteins (CD37mfEC2-FcHis and CD37EC2-FcHis) were purified from cell culture supernatants using protein A affinity chromatography.

Immunization of rabbits
Immunization of rabbits was performed at MAB Discovery GMBH (Neuried, Germany). Rabbits were repeatedly immunized with a mixture of CD37EC2-FcHis and CD37mfEC2-FcHis or HEK293F cells transiently expressing human or mfCD37. Blood of these rabbits was collected and B lymphocytes were isolated. Single B cells were sorted into wells of microtiter plates using a proprietary process of MAB Discovery and further expanded. Supernatants of these single B cells were analyzed for specific binding to CHO-S cells transiently expressing CD37 (CHO-CD37) and CHO-S cells transiently expressing mfCD37 (CHO-mfCD37).

Recombinant antibody production Primary screening results were analyzed and primary hits were selected for sequencing, recombinant mAb production and purification. Unique variable heavy (VH) and light (VL) chain coding regions were gene synthesized and cloned into mammalian expression vectors containing human IgG1 constant region coding sequences (IgG1 allotype G1m(f) with Ig kappa chain and E430G mutation (EU numbering) heavy chain). During this process, undesired unpaired cysteines in some antibody light chains were replaced by serine.

Recombinant chimeric antibodies were produced in HEK293 cells by transiently co-transfecting heavy chain (HC) and light chain (LC) encoding expression vectors using an automated approach on a Tecan Freedom Evo platform. Immunoglobulins were purified from cell supernatants using affinity purification (Protein A) on a Dionex Ultimate 3000 HPLC system.

The reactivity of the produced chimeric (VH rabbit, Fc human) monoclonal antibodies (mAbs) containing the mutation E430G was analyzed again for binding to CHO-CD37 or CHO-mfCD37 cells. In addition, binding to the human lymphoma cell line Daudi and functionality in a CDC assay on Daudi cells were analyzed.

Example 2: Humanization of a rabbit chimeric antibody
Generation of humanized antibody sequences
Humanized antibody sequences were generated from rabbit antibodies, rabbit anti-CD37-004, -005, -010 and -016 at Antitope (Cambridge, UK). Humanized antibody sequences were generated using germline humanization (CDR grafting) technology. Humanized V-region genes were designed based on human germline sequences with maximum homology to the VH and Vκ amino acid sequences of rabbit and mouse antibodies. For each rabbit antibody, a set of four to six VH and a set of four or five Vκ (VL) germline humanized V-region genes were designed.

A structural model of a rabbit antibody V-region was generated using the Swiss PDB and analyzed to identify amino acids in the V-region framework that may be important for antibody binding. These amino acids were noted for incorporation into one or more variant CDR-grafted antibodies.

The heavy and light chain V region amino acid sequences were compared against a database of human germline V and J segment sequences to identify heavy and light chain human sequences with the greatest degree of homology for use as human variable domain frameworks. The germline sequences used as the basis for the humanization design are shown in Table 2.

Table 2. Maximally Matched Human Germline V and J Segment Sequences

A series of humanized heavy and light chain V regions were then designed by grafting the CDRs onto the framework and, if necessary, backmutating residues that may be important for antibody binding (identified by structural modeling) to rabbit residues. The variant sequences with the lowest occurrence of potential T cell epitopes were then selected using Antitope's proprietary in silico technology iTope™ and TCED™ (T Cell Epitope Database) (Perry, L.C.A, Jones, T.D. and Baker, M.P. New Approaches to Prediction of Immune Responses to Therapeutic Proteins during Preclinical Development (2008). Drugs in R&D 9(6): 385-396; Bryson, C.J., Jones, T.D. and Baker, M.P. Prediction of Immunogenicity of Therapeutic Proteins (2010). Biodrugs 24(1):1-8). Finally, the nucleotide sequences of the designed variants were codon-optimized.

For the antibody IgG1-016-H5L2, a variant was generated with a point mutation in the variable domain to replace the free cysteine: IgG1-016-H5L2-LC90S (also with the additional mutations F405L and E430G). This variant was generated by gene synthesis (Geneart).

The variable region sequences of the humanized CD37 antibodies are shown in the sequence listing herein and in Table 1 above.

Example 3: Generation of bispecific antibodies Bispecific IgG1 antibodies were generated by Fab arm exchange under controlled reducing conditions.The basis of this method is the use of complementary CH3 domains to promote the formation of heterodimers under specific assay conditions described in WO2011/131746.F405L and K409R (EU numbering) mutations were introduced into CD37 antibody to create antibody pairs with complementary CH3 domains.In a specific case, F405L and K409R were combined with E430G mutation.

To generate bispecific antibodies, the two parental complementary antibodies (each at a final concentration of 0.5 mg/mL) were incubated with 75 mM 2-mercaptoethylamine HCl (2-MEA) in a total volume of 100 μL TE for 5 h at 31 °C. The reduction reaction was stopped by removing the reducing agent 2-MEA using a spin column (Microcon centrifugal filter, 30k, Millipore) according to the manufacturer's protocol.

Example 4: Expression constructs for antibodies, transient expression and purification For antibody expression, the VH and VL sequences were cloned into an expression vector (pcDNA3.3) containing the appropriate constant heavy chain (HC) region in the case of VH, in certain cases the F405L or K409R mutation and/or the E345R or E430G mutation, and the light chain (LC) region in the case of VL.

The antibodies were expressed as IgG1κ. Plasmid DNA mixtures encoding both the heavy and light chains of the antibodies were transiently transfected into Expi293F cells (Life technologies, USA) using 293fectin (Life technologies) essentially as described by Vink et al. (Vink et al., Methods, 65(1), 5-10 2014). The antibodies were then purified by immobilized protein G chromatography.

The following antibodies were used in the examples:

Wild-type IgG1 antibodies:
IgG1-004-H5L2 (having the VH and VL sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 5)
IgG1-005-H1L2 (having the VH and VL sequences set forth in SEQ ID NO: 8 and SEQ ID NO: 12)
IgG1-010-H5L2 (having the VH and VL sequences set forth in SEQ ID NO: 15 and SEQ ID NO: 19)
IgG1-016-H5L2 (having the VH and VL sequences set forth in SEQ ID NO: 22 and SEQ ID NO: 26)
IgG1-G28.1 (having VH and VL sequences as set forth in SEQ ID NO: 39 and SEQ ID NO: 43 - based on SEQ ID NOs: 1 and 3 of EP2241577)
IgG1-G28.1-K409R-delK (also contains the C-terminal heavy chain mutation 445-PG-446)
IgG1-37.3 (having VH and VL sequences as set forth in SEQ ID NO: 46 and SEQ ID NO: 50 - based on SEQ ID NOs: 55 and 72 of WO2011/112978)
IgG1-b12 (having the VH and VL sequences set forth in SEQ ID NO: 32 and SEQ ID NO: 36 - based on the gp120-specific antibody b12 [Barbas, CF. J Mol Biol. 1993 Apr 5;230(3):812-23])

IgG1 antibody with Fc-Fc interaction enhancing mutation E430G:
IgG1-004-H5L2-E430G
IgG1-005-H1L2-E430G
IgG1-010-H5L2-E430G
IgG1-016-H5L2-E430G
IgG1-G28.1-E430G
IgG1-37.3-E430G
IgG1-b12-E430G
IgG1-005-H1L2-K409R-E430G
IgG1-010-H5L2-K409R-E430G
IgG1-016-H5L2-F405L-E430G
IgG1-016-H5L2-LC90S-F405L-E430G
IgG1-004-E430G
IgG1-005-E430G
IgG1-010-E430G
IgG1-016-E430G

IgG1 antibody with the Fc-Fc interaction enhancing mutation E430S:
IgG1-010-H5L2-K409R-E430S
IgG1-016-H5L2-F405L-E430S

IgG1 antibody with Fc-Fc interaction enhancing mutation E345K:
IgG1-010-H5L2-K409R-E345K
IgG1-016-H5L2-F405L-E345K

IgG1 antibody with Fc-Fc interaction enhancing mutation E345R:
IgG1-G28.1-E345R
IgG1-b12-E345R
IgG1-010-H5L2-K409R-E345R
IgG1-016-H5L2-F405L-E345R

Bispecific antibodies
bsIgG1-016-H5L2-F405L×IgG1-005-H1L2-K409R
bsIgG1-016-H5L2-F405L×IgG1-010-H5L2-K409R

Bispecific antibody with Fc-Fc interaction enhancing mutation E430G:
bsIgG1-016-H5L2-LC90S-F405L-E430G×005-H1L2-K409R-E430G
bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G
bsIgG1-016-H5L2-LC90S-F405L-E430G×b12-K409R-E430G
bsIgG1-b12-F405L-E430G×010-H5L2-K409R-E430G
bsIgG1-b12-F405L-E430G×005-H1L2-K409R-E430G

IgG1 antibody with the FcγR interaction enhancing mutation S239D-I332E:
IgG1-G28.1-S239D-I332E

Example 5: Introduction of Fc-Fc interaction enhancing mutations into CD37 antibodies results in a new enhanced ability to induce complement dependent cytotoxicity (CDC)
Determination of complement-dependent cytotoxicity (CDC)
In the first experiment, tumor cells from untreated CLL patients (AllCells, California, USA) were resuspended in RPMI containing 0.2% BSA (bovine serum albumin) and seeded at a density of ^0.2x105 cells/well (40μL/well) in polystyrene 96-well round-bottom plates (Greiner bio-one Cat # 650101) and 40μL of a concentration series of IgG1-G28.1-K409R-delK, IgG1-G28.1-E345R or IgG1-b12-E345R (0.003-10μg/mL final antibody concentration) was added. IgG1-b12-E345R (based on the gp120-specific antibody b12 [Barbas, CF. J Mol Biol. 1993 Apr 5;230(3):812-23]) was used as a negative control. It should be noted that in the case of IgG1-G28.1-K409R-delK, the K409R mutation has no effect on the binding or CDC-inducing capacity.Similarly, the delK(445-PG-446) mutation, which was introduced into the antibody to facilitate biochemical analysis, did not affect the target-binding or CDC-inducing capacity (see below).

After incubation (10 min at room temperature with shaking), 20 μL of pooled normal human serum (NHS Cat # M0008 Sanquin, Amsterdam, The Netherlands) was added to each well as a complement source, and the plate was incubated at 37°C for 45 min. The reaction was stopped by chilling the plate on ice. Propidium iodide (PI; 10 μL of a 10 μg/mL solution; Sigma-Aldrich Chemie B.V., Zwijndrecht, The Netherlands) was then added, and lysis was detected by measuring the percentage of dead cells (corresponding to PI-positive cells) by flow cytometry (FACS Canto II; BD Biosciences). Graphs were generated using best-fit values of a nonlinear dose-response fit with log-transformed concentrations in GraphPad Prism V6.04 software (GraphPad Software, San Diego, CA, USA).

In a second experiment, tumor cells from another untreated CLL patient (AllCells, California, USA) were resuspended in RPMI containing 0.2% BSA and seeded at a density of ^0.5x105 cells/well (30μL/well) into polystyrene 96-well round-bottom plates (Greiner bio-one Cat # 650101) and 50μL of a concentration series of IgG1-G28.1, IgG1-G28.1-E430G or IgG1-b12 (0.003-10μg/mL final antibody concentration in 3.33x serial dilutions) was added. After incubation (15 min at room temperature), 20μL of pooled normal human serum (NHS Cat # M0008 Sanquin, Amsterdam, The Netherlands) was added to each well as a complement source and the plates were incubated at 37°C for 45 min. The reaction was stopped by cooling the plates on ice. Propidium iodide (PI; 20 μL of a 10 μg/mL solution; Sigma-Aldrich Chemie BV, Zwijndrecht, The Netherlands) was then added and lysis was detected by measuring the percentage of dead cells (corresponding to PI-positive cells) by flow cytometry (FACS Canto II; BD Biosciences). Graphs were generated using best-fit values from a nonlinear dose-response fit with log-transformed concentrations in GraphPad Prism V6.04 software (GraphPad Software, San Diego, CA, USA).

Figures 1A and B show that the CD37 antibody G28.1 without the Fc-Fc interaction-enhancing E345R or E430G mutations (IgG1-G28.1 or IgG1-G28.1-K409R-delK) did not induce CDC in primary tumor cells from CLL patients, whereas G28.1 with the Fc-Fc interaction-enhancing mutations E345R or E430G (IgG1-G28.1-E345R or IgG1-G28.1-E430G) induced profound dose-dependent CDC in primary CLL cells.

Quantitative measurement of cell surface antigens by flow cytometry (Qifi)
The Human IgG Calibrator Kit (Biocytix Cat # CP010) was used to measure CD37 and membrane complement regulatory protein (mCRP; CD46, CD55, and CD59) expression levels on CLL tumor cells. Briefly, tumor cells from CLL patients (as in the first experiment above) resuspended in RPMI containing 0.2% BSA were seeded into polystyrene 96-well round-bottom plates (Greiner bio-one Cat # 650101) at a density of 0.5 x ¹⁰⁵ cells/well (30 μL/well), centrifuged, and 50 μL of CD37 (Abcam, cat. no. 76522) or control mouse antibody (Purified Mouse IgG1κ Isotype Control, Clone MOPC-21; BD cat. no. 555746) was added. After incubation (30 min at 4°C), 50 μL of calibration beads were added to separate wells. After washing the beads and cells twice (150 μL FACS buffer, centrifugation at 300×g at 4° C. for 3 min between washing steps), 50 μL/well of the secondary antibody (FITC conjugate) dilution provided in the Human IgG Calibrator Kit was added. After incubation in the dark (4° C. for 45 min), the cells were washed twice with FACS buffer, resuspended in 35 μL FACS buffer, and analyzed by flow cytometry (Intellicyt iQue™ Screener). The amount of antigen was determined by calculating the antibody binding capacity based on the calibration curve according to the manufacturer's guidelines.

Figure 2 shows that CD37 was highly expressed in primary tumor cells from this CLL patient. The patient showed normal mCRP expression levels.

Example 6: Binding of CD37 antibodies and variants to cell surface expressed CD37 Binding to cell surface expressed CD37 (Daudi cells, CHO cells expressing cynomolgus monkey CD37) was measured by flow cytometry. Cells resuspended in RPMI containing 0.2% BSA were plated at 100,000 cells/well in polystyrene 96-well round bottom plates (Greiner bio-one Cat # 650101) and centrifuged at 300×g for 3 minutes at 4° C. Serial dilutions of CD37 or control antibodies (0.003-10 μg/mL final antibody concentration in 3.33× serial dilutions) were added and cells were incubated for 30 minutes at 4° C. Plates were washed twice with FACS buffer (PBS/0.1% BSA/0.01% sodium azide) and centrifuged. The cells were then incubated with R-phycoerythrin (PE)-conjugated goat anti-human IgG F(ab')2 (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA; cat #: 109-116-098) diluted 1/100 in PBS/0.1% BSA/0.01% sodium azide for 30 min at 4°C. The cells were washed/spun twice using FACS buffer, resuspended in 30 μL FACS buffer, and analyzed by measuring mean fluorescence intensity using an Intellicyt iQue™ screener (Westburg). Binding curves were generated using nonlinear regression (sigmoidal dose-response, variable slope) analysis within GraphPad Prism V6.04 software (GraphPad Software, San Diego, CA, USA).

Binding to Daudi cells Figure 3 shows that the humanized CD37 antibodies IgG1-004-H5L2, IgG1-005-H1L2, IgG1-010-H5L2 and IgG1-016-H5L2 showed dose-dependent binding to Daudi cells. Introduction of the Fc-Fc interaction enhancing E430G mutation into these antibodies and, in the case of IgG1-005-H1L2, additionally the K409R mutation did not affect binding.

Figure 4 shows that introduction of the E430G mutation into IgG1-G28.1 or IgG1-37.3 did not affect binding to Daudi cells.

For antibody IgG1-016-H5L2, a variant was generated with a point mutation in the variable domain to replace a free cysteine in the light chain: IgG1-016-H5L2-LC90S. This variant was also generated with the additional F405L and E430G mutations, which have previously been shown not to affect target binding properties. Figure 5 shows that IgG1-016-H5L2, IgG1-016-H5L2-E430G, IgG1-016-H5L2-F405L-E430G and IgG1-016-H5L2-LC90S-F405L-E430G all showed comparable binding to Daudi cells, thus indicating that the LC90S mutation did not affect binding.

Binding to CHO cells expressing cynomolgus monkey CD37 Binding to CHO cells expressing cynomolgus monkey CD37 was measured by flow cytometry using the method described above. Figure 6 shows that IgG1-004-H5L2-E430G, IgG1-005-H1L2-E430G, IgG1-010-H5L2-E430G and IgG1-016-H5L2-E430G showed dose-dependent binding to CHO cells expressing cynomolgus monkey CD37. IgG1-G28.1 and IgG1-28.1-E430G did not bind to CHO cells expressing cynomolgus monkey CD37.

Example 7: Identification of CD37 antibodies that do not compete for binding to CD37
(Lack of) binding competition – measured by flow cytometry
CD37 antibody was labeled with Alexa Fluor 488 NHS Ester (succinimidyl ester). 1 mg of CD37 antibody (dissolved in PBS) was transferred to a 1 ml microcentrifuge vial (reaction vial). The pH was increased by adding 10% by volume of 1 M sodium bicarbonate buffer (pH 9). Immediately prior to use, 1 mg of Alexa Fluor 488 NHS Ester (adjusted to room temperature) was dissolved in 100 μL of DMSO. The labeling reaction was initiated by the addition of 10 μL of fresh Alexa dye solution per mg of antibody. The reaction vials were capped and mixed gently by inversion. After 1 h of incubation at room temperature, the reactions were quenched by adding 50 μL of 1 M Tris to each reaction vial. Unreacted dye was removed from the Alexa-labeled antibodies by gel filtration using a BioRad PDP10 column equilibrated with borate-buffered saline according to the manufacturer's instructions. The Alexa-labeled antibodies were stored at 4°C and protected from light.

Binding competition between different CD37 antibodies was measured by flow cytometry. Raji cells (ATCC, CCL-86) were resuspended in Raji medium (RPMI 1640, 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 10 mM HEPES, and 1 mM pyruvate) at a concentration of 1× ¹⁰⁷ cells/mL. A 30 μL aliquot of the cell suspension was then transferred to a FACS tube with a 30 μL aliquot of unlabeled antibody solution (40 μg/mL final concentration). The mixture was incubated at 37° C. for 15 min with gentle shaking. A488 labeled antibody dilutions were then prepared, and after incubation, 10 μL of labeled antibody (4 μg/mL final concentration) was transferred to the FACS tube containing unlabeled antibody and cells. The mixture was incubated at 37° C. for 15 min with gentle shaking. After incubation, samples were quenched by adding 4 ml of ice-cold PBS, centrifuged at 2000 rpm for 3 min at 4°C, aspirated twice, and then resuspended in 125 μL of PBS. Binding competition was analyzed by measuring mean fluorescence intensity using a BD FACSCalibur (BD Biosciences). For quantification, fluorescence intensity was converted to MESF (molecule of soluble fluorescence).

Figures 7A and 8 show that preincubation of Raji cells with IgG1-005-H1L2-E430G and IgG1-010-H5L2-E430G blocked subsequent binding of IgG1-005-H1L2-E430G and IgG1-010-H5L2E430G, but did not block subsequent binding of IgG1-37.3-E430G, IgG1-G28.1-E430G, IgG1-004-H5L2-E430G, and IgG1-016-H5L2-E430G.

Preincubation of Raji cells with IgG1-004-H5L2-E430G substantially reduced the subsequent binding of IgG1-37.3-E430G, IgG1-G28.1-E430G, IgG1-004-H5L2-E430G, and IgG1-016-H5L2-E430G, but did not reduce the subsequent binding of IgG1-005-H1L2-E430G, and IgG1-010-H5L2-E430G.

Preincubation of Raji cells with IgG1-016-H5L2-E430G blocked subsequent binding of IgG1-37.3-E430G, IgG1-G28.1-E430G, IgG1-004-H5L2-E430G, and IgG1-016-H5L2-E430G, but did not block subsequent binding of IgG1-005-H1L2-E430G and IgG1-010-H5L2-E430G.

Preincubation of cells with IgG1-37.3-E430G blocked subsequent binding of all antibodies tested. However, as shown above, preincubation with either IgG1-005-H1L2-E430G or IgG1-010-H5L2-E430G did not block binding of IgG1-37.3-E430G.

Preincubation of cells with IgG1-G28.1-E430G blocked subsequent binding of IgG1-37.3-E430G, IgG1-G28.1-E430G, IgG1-004-H5L2-E430G, and IgG1-016-H5L2-E430G, but did not block subsequent binding of IgG1-005-H1L2-E430G and IgG1-010-H5L2-E430G.

(Lack of) Binding Competition - Measured by Functional Screening Using CDC Assays To determine whether non-cross-blocking CD37 antibodies exhibit enhanced CDC when combined and to confirm the feasibility of functionally combining non-cross-blocking CD37 antibodies, CDC assays were performed using individual CD37 antibodies and their combinations.

Raji cells, resuspended in RPMI containing 0.2% BSA, were seeded in polystyrene 96-well round-bottom plates (Greiner bio-one Cat # 650101) at a density of ^1x105 cells/well (30μL/well) and 50μL of humanized CD37 antibodies, their variants, their combinations, or the control antibody IgG1-b12 was added (final antibody concentration 10μg/mL, combination: 5+5μg/mL). After incubation (15 min at room temperature with shaking), 20μL of pooled normal human serum (NHS Cat # M0008 Sanquin, Amsterdam, The Netherlands) was added to each well and the plates were incubated at 37°C for 45 min. The plates were centrifuged (3 min, 1200 rpm) and the supernatant was discarded. Propidium iodide (PI; 30 μL of a 1.67 μg/mL solution; Sigma-Aldrich Chemie BV, Zwijndrecht, The Netherlands) was added and lysis was detected by measuring the percentage of dead cells (corresponding to PI-positive cells) by flow cytometry (Intellicyt iQue™ screener, Westburg). Data were analyzed using GraphPad Prism software (GraphPad Software, San Diego, CA, USA).

Figures 7B and C show that the combinations IgG1-004-H5L2 + IgG1-010-H5L2 (with or without the E430G mutation) and IgG1-005-H1L2 + IgG1-016-H5L2 (with or without the E430G mutation) induced enhanced CDC compared to their individual counterparts. The combinations IgG1-004-H5L2 + IgG1-016-H5L2 (with or without the E430G mutation) did not induce enhanced CDC compared to their individual counterparts.

Figures 7D and E show that the combinations IgG1-004-H5L2 + IgG1-005-H1L2 (with or without the E430G mutation) and IgG1-010-H5L2 + IgG1-016-H5L2 (with or without the E430G mutation) induced enhanced CDC compared to their individual counterparts. The combinations IgG1-005-H1L2 + IgG1-010-H5L2 (with or without the E430G mutation) did not induce enhanced CDC compared to their individual counterparts.

Figures 7F and G show that the combinations IgG1-37.3 + IgG1-005-H1L2 (with or without the E430G mutation) and IgG1-37.3 + IgG1-010-H5L2 (with or without the E430G mutation) induced enhanced CDC compared to their individual counterparts.

Thus, functional combination studies confirmed the results of the described binding competition studies with CD37 and demonstrated that non-cross-blocking CD37 antibodies can be functionally combined.

Example 8: Introduction of Fc-Fc interaction enhancing mutations into a humanized CD37 antibody results in a new enhanced ability to induce complement dependent cytotoxicity (CDC)
Daudi cells, resuspended in RPMI containing 0.2% BSA, were seeded into polystyrene 96-well round-bottom plates (Greiner bio-one Cat # 650101) at a density of 1 x ¹⁰⁵ cells/well (30 μL/well) and 50 μL of a concentration series of humanized CD37 antibodies and their variants or the control antibody IgG1-b12 (0.003-10 μg/mL final antibody concentration in 3.33x serial dilutions) was added. After incubation (15 min at room temperature), 20 μL of pooled normal human serum (NHS Cat # M0008 Sanquin, Amsterdam, The Netherlands) was added to each well and the plates were incubated at 37°C for 45 min. The plates were centrifuged (3 min, 1200 rpm) and the supernatant was discarded. Propidium iodide (PI; 30 μL of a 1.67 μg/mL solution; Sigma-Aldrich Chemie BV, Zwijndrecht, The Netherlands) was added and lysis was detected by measuring the percentage of dead cells (corresponding to PI-positive cells) by flow cytometry (Intellicyt iQue™ screener, Westburg). Graphs were generated using best-fit values from a nonlinear dose-response fit with log-transformed concentrations in GraphPad Prism V6.04 software (GraphPad Software, San Diego, CA, USA).

Figure 9 shows that IgG1-004-H5L2, IgG1-005-H1L2, IgG1-010-H5L2 and IgG1-016-H5L2 did not induce CDC in Daudi cells. Upon introduction of the Fc-Fc interaction-enhancing E430G mutation, these antibodies (IgG1-004-H5L2-E430G, IgG1-005-H1L2-E430G, IgG1-010-H5L2-E430G and IgG1-016-H5L2-E430G) induced profound dose-dependent CDC in Daudi cells.

Figure 10A shows that IgG1-G28.1 and IgG1-37.3 did not induce CDC in Daudi cells. When the Fc-Fc interaction-enhancing E430G mutation was introduced, these antibodies (IgG1-G28.1-E430G and IgG1-37.3-E430G) induced profound dose-dependent CDC in Daudi cells.

For antibody IgG1-016-H5L2, a variant with a point mutation in the variable domain to replace a free cysteine in the light chain was generated: IgG1-016-H5L2-LC90S. In addition, this variant was also generated with the F405L mutation (previously shown not to affect target binding or CDC) and the Fc-Fc interaction enhancing E430G mutation. Figure 11 shows that IgG1-016-H5L2-E430G, IgG1-016-H5L2-F405L-E430G and IgG1-016-H5L2-LC90S-F405L-E430G all showed comparable activity in the in vitro CDC assay, thus indicating that the LC90S mutation did not affect the ability to induce CDC. IgG1-016-H5L2 did not induce CDC in Daudi cells.

Introduction of other Fc-Fc interaction enhancing mutations E345K, E345R, E430S and RRGY into IgG1-010-H5L2 and IgG1-016-H5L2 also caused profound CDC in Daudi cells. Figures 10B and C show that the maximal lysis of Daudi cells was comparable for all Fc-Fc interaction enhancing mutations tested.

Example 9: Bispecific CD37 antibodies with Fc-Fc interaction enhancing mutations are more potent at inducing CDC than monospecific bivalent CD37 antibodies with Fc-Fc interaction enhancing mutations due to monovalent binding and dual epitope targeting
The F405L or K409R mutation was introduced into a humanized CD37 antibody containing an E430G mutation to allow the generation of a bispecific antibody (bsIgG1) with two CD37-specific Fab arms that do not compete for binding to CD37. The ability of the bispecific CD37 antibody containing an E430G mutation to induce CDC was measured as described above and compared to the ability of a CD37 monospecific bivalent antibody containing an E430G mutation, a combination of two CD37 monospecific bivalent antibodies containing an E430G mutation that do not compete for binding to CD37 (the final concentration of the combined antibodies is the same as the concentration of the individual bispecific antibodies), a monovalent CD37 antibody containing an E430G mutation (i.e. a bispecific antibody that contains one CD37-specific Fab arm and one non-binding Fab arm derived from IgG1-b12 and contains an E430G mutation) or a combination of two monovalent CD37 antibodies containing an E430G mutation that do not compete for binding to CD37.

CDC in Daudi cells
Figure 12A shows that bsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G was more potent than either IgG1-005-H1L2-E430G or IgG1-016-H5L2-E430G in inducing CDC in Daudi cells. The bispecific bsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G was also more potent than the combination of IgG1-005-H1L2-K409R-E430G + IgG1-016-H5L2-F405L-E430G. The monovalent CD37-binding antibodies bsIgG1-b12-F405L-E430Gx005-H1L2-K409R-E430G and bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G also induced CDC in Daudi cells, but less efficiently than bsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G.

Figure 12B shows that bsIgG1-016-H5L2-LC90S-F405L-E430G x 010-H5L2-K409R-E430G was more potent than either IgG1-010-H5L2-E430G or IgG1-016-H5L2-E430G in inducing CDC in Daudi cells. The bispecific bsIgG1-016-H5L2-LC90S-F405L-E430G x 010-H5L2-K409R-E430G was also more potent than the combination of IgG1-010-H5L2-E430G + IgG1-016-H5L2-E430G. The monovalent binding antibodies bsIgG1-016-H5L2-LC90S-F405L-E430G×b12-K409R-E430G and bsIgG1-b12-F405L-E430G×010-H5L2-K409R-E430G also induced CDC in Daudi cells, whereas bsIgG1-b12-F405L-E430G×010-H5L2-K409R-E430G was significantly more potent than bsIgG1-016-H5L2-LC90S-F405L-E430G×b12-K409R-E430G. It was less potent than 6-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G, and bsIgG1-016-H5L2-LC90S-F405L-E430G×b12-K409R-E430G was equally potent as bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G.

We also compared the ability of bispecific CD37 antibodies containing the E430G mutation to induce CDC with that of bispecific CD37 antibodies without the E430G mutation. FIG. 13 shows that bsIgG1-016-H5L2-F405L×005-H1L2-K409R and bsIgG1-016-H5L2-F405L×010-H5L2-K409R were able to induce CDC in Daudi cells, but were less potent at doing so compared to their E430G-containing counterparts bsIgG1-016-H5L2-LC90S-F405L-E430G×005-H1L2-K409R-E430G and bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G.

CDC in OCI-Ly-7 cells
FIG. 12C shows that the monovalent binding antibodies bsIgG1-016-H5L2-LC90S-F405L-E430G×b12-K409R-E430G and bsIgG1-b12-F405L-E430G×010-H5L2-K409R-E430G were more potent in inducing CDC in OCI-Ly-7 cells compared to their monospecific bivalent binding counterparts IgG1-016-H5L2-E430G and IgG1-010-H5L2-E430G. The monovalent binding antibody (bsIgG1-016-H5L2-LC90S-F405L-E430G x b12-K409R-E430G + bsIgG1-b12-F405L-E430G x 010-H5L2-K409R-E430G) was more potent than the bivalent antibody combination (IgG1-010-H5L2-E430G + IgG1-016-H5L2-E430G), as demonstrated by consistently lower EC50s in two independent experiments (Figure 12D). Furthermore, bsIgG1-016-H5L2-LC90S-F405L-E430G × 010-H5L2-K409R-E430G was more potent than the bivalent antibody combination (IgG1-010-H5L2-E430G + IgG1-016-H5L2-E430G) in inducing CDC in OCI-Ly-7 cells, as demonstrated by consistently lower EC50s in three independent experiments (Figure 12E).

The ability of the monovalent binding antibody combination (bsIgG1-016-H5L2-LC90S-F405L-E430G x b12-K409R-E430G + bsIgG1-b12-F405L-E430G x 010-H5L2-K409R-E430G) to induce CDC in OCI-Ly-7 cells was comparable to that of bsIgG1-016-H5L2-LC90S-F405L-E430G x 010-H5L2-K409R-E430G.

CDC in primary CLL tumor cells
The ability of bispecific CD37 antibodies containing the E430G mutation to induce CDC in tumor cells from CLL patients was measured as described above and compared with the ability of CD37 antibodies containing the E430G mutation or combinations of CD37 antibodies containing the E430G mutation or monovalent CD37 antibodies containing the E430G mutation.

Figure 14A shows that bsIgG1-016-H5L2-LC90S-F405L-E430G×005-H1L2-K409R-E430G was more potent than either IgG1-005-H1L2-K409R-E430G or IgG1-016-H5L2-F405L-E430G in inducing CDC in primary CLL tumor cells. The bispecific bsIgG1-016-H5L2-LC90S-F405L-E430G×005-H1L2-K409R-E430G was also more potent than the combination of IgG1-005-H1L2-K409R-E430G + IgG1-016-H5L2-F405L-E430G. The monovalent binding antibodies bsIgG1-b12-F405L-E430G×005-H1L2-K409R-E430G and bsIgG1-016-H5L2-LC90S-F405L-E430G×b12-K409R-E430G also induced CDC in primary CLL tumor cells, but less efficiently than bsIgG1-016-H5L2-LC90S-F405L-E430G×005-H1L2-K409R-E430G.

Figure 14B shows that bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G was more potent than either IgG1-010-H5L2-E430G or IgG1-016-H5L2-E430G in inducing CDC in primary CLL tumor cells. The bispecific bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G was also more potent than the combination of IgG1-010-H5L2-E430G + IgG1-016-H5L2-E430G. The monovalent binding antibodies bsIgG1-016-H5L2-LC90S-F405L-E430G×b12-K409R-E430G and bsIgG1-b12-F405L-E430G×010-H5L2-K409R-E430G also induced CDC in primary CLL tumor cells, whereas bsIgG1-b12-F405L-E430G×010-H5L2-K409R-E430G was significantly lower than bsIgG1-0 It was less potent than 16-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G, and bsIgG1-016-H5L2-LC90S-F405L-E430G×b12-K409R-E430G was equally potent as bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G.

Example 10: Bispecific CD37 antibodies with Fc-Fc interaction enhancing mutations induce CDC in various B cell lymphoma cell lines exhibiting a broad range of CD37 expression The ability of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G to induce CDC at a concentration of 10 μg/mL in various B cell lymphoma cell lines derived from various B cell lymphoma subtypes was measured (as described above). The expression levels of CD37 molecules on the cell surface of these cell lines were measured by quantitative flow cytometry as described above.

Table 3 provides an overview of the cell lines tested.

Table 3. B cell lymphoma cell lines

Figure 15 shows that bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G induced CDC in a broad range of B cell lymphoma cell lines derived from different B cell lymphoma types.

Example 11: Bispecific CD37 antibodies with Fc-Fc interaction enhancing mutations are more potent in inducing antibody-dependent cellular cytotoxicity (ADCC)
Labeling of target cells
The ability of CD37 antibodies to induce ADCC was measured by chromium release assay. Daudi or Raji cells were harvested (5x106 cells/mL) in 1mL of medium (RPMI 1640 supplemented with 10% donor bovine serum with iron (DBSI; ThermoFischer, Cat # 10371029) and penicillin- ^streptomycin mixture (pen/strep) plus 100 μCi of 51Cr (Chromium-51; PerkinElmer, Cat # NEZ030005MC). Cells were incubated for 1 hour with shaking in a 37°C water bath. After washing the cells (twice in PBS, 1500 rpm, 5 min), the cells were resuspended in RPMI 1640/10% DBSI/pen/strep and counted by trypan blue exclusion test. Cells were diluted to a density of ^1x105 cells/mL.

Preparation of effector cells. Peripheral blood mononuclear cells (Sanquin, Amsterdam, The Netherlands) from healthy volunteers were isolated from 45 mL of freshly drawn heparinized blood (buffy coat) by Ficoll density gradient centrifugation (Bio Whittaker; Lymphocyte Separation Medium, cat 17-829E) according to the manufacturer's instructions. Cells were resuspended in RPMI 1640/10% DBSI/pen/strep, counted by trypan blue exclusion, and diluted to a density of 1 x ¹⁰⁷ cells/mL.

ADCC assay procedure
Fifty microliters of ^51Cr -labeled target cells were pipetted into a 96-well round-bottom microtiter plate (Greiner Bio-One; Cat # 650101) and 50 μL of a concentration series of CD37 or control antibodies (1.5-5,000 ng/mL final concentration in 3-fold dilutions) in RPMI 1640/10% DBSI/pen/strep was added. Cells were incubated for 15 min at room temperature (RT) and 50 μL of effector cells were added to yield an effector:target ratio of 100:1. Cells were incubated for 4 h at 37°C and 5% CO2. For the measurement of maximum lysis, 50 μL (5.000 cells) of 51Cr-labeled Daudi cells were incubated with 100 μL of 5% Triton-X100; for the measurement of spontaneous lysis (background lysis), 5,000 51Cr-labeled Daudi cells were incubated in 150 μL of medium without any antibody or effector cells. The level of antibody-independent cell lysis was measured by incubating 5,000 Daudi cells with 500,000 PBMCs without antibody. The plates were centrifuged (1200 rpm, 10 min) and 25 μL of the supernatant was transferred to 100 μL of Microscint-40 solution (Packard, Cat # 6013641) in a 96-well plate. The plates were sealed and shaken at 800 rpm for 15 min, and the released 51Cr was counted by a scintillation counter (TopCount®, PerkinElmer). The specific lysis percentage was calculated as follows:
% specific lysis=(sample cpm−spontaneous lysis cpm)/(maximal lysis cpm−spontaneous lysis cpm), where cpm is counts per minute.

Figure 16A shows that bsIgG1-016-H5L2-LC90S-F405L-E430G x 005-H1L2-K409R-E430G was more potent than either IgG1-005-H1L2-K409R-E430G or IgG1-016-H5L2-F409L-E430G or the combination of IgG1-005-H1L2-K409R-E430G + IgG1-016-H5L2-F405L-E430G in inducing ADCC in Daudi cells.

Figure 16B shows that bsIgG1-016-H5L2-LC90S-F405L-E430G × 010-H5L2-K409R-E430G was more potent than either IgG1-010-H5L2-E430G or IgG1-016-H5L2-E430G or the combination of IgG1-010-H5L2-E430G + IgG1-016-H5L2-E430G in inducing ADCC in Daudi cells.

Figure 16C shows similar results to Figure 16B for PBMCs from different donors and additionally shows that bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G was more potent in inducing ADCC in Raji cells than the monovalent binding antibodies bsIgG1-016-H5L2-LC90S-F405L-E430G×b12-K409R-E430G and bsIgG1-b12-F405L-E430G×010-H5L2-K409R-E430G.

Example 12: Bispecific CD37 antibodies with Fc-Fc interaction enhancing mutations induce potent ex vivo CDC in primary tumor cells derived from patients with various B cell malignancies
The CDC potency of bsIgG1-016-H5L2-LC90S-F405L×010-H5L2-K409R-E430G was analyzed using patient-derived primary tumor cells from five different B-cell malignancies: chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), and non-Hodgkin's lymphoma (not further specified). All patient samples were obtained and stored after written informed consent, using protocols approved by the VUmc Medical Ethics Committee, in accordance with the Declaration of Helsinki. Patient bone marrow mononuclear cells (BMNCs) or peripheral blood mononuclear cells (PBMCs) were isolated from patients' bone marrow aspirates or peripheral blood samples by density gradient centrifugation (Ficoll-Paque PLUS, GE Healthcare). Cells were used immediately or stored in liquid nitrogen until further use.

Patient lymph node tissue was cut into small pieces and collected in α-MEM medium (ThermoFischer Scientific, Waltham, MA) containing 1% penicillin-streptomycin, 0.2% heparin, and 5% platelet lysate, and left at 37°C overnight. After incubation, the supernatant (non-stromal cell compartment containing tumor cells) was collected, and cells were strained using a 70 μM Easy Strainer (Greiner Bio-one). Cells were counted, resuspended in RPMI1640 medium containing 25% heat-inactivated FBS and 10% DMSO, and frozen in liquid nitrogen until further use.

The QifiKit (DAKO, cat. no. K007811) was used to measure CD37 and membrane complement regulatory protein (mCRP; CD46, CD55 and CD59) expression levels on isolated patient cells. Cells were incubated with purified antibodies CD37 (BD, cat. no. 555456), CD46 (BioLegend, cat. no. 352404), CD55 (BioLegend, cat. no. 311302), CD59 (BioLegend, cat. no. 304702) and b12 (Genmab) for 30 min at 4°C. After this, the method provided by the QifiKit manufacturer was used. After the final step of the Qifi Kit procedure, cells were incubated with lymphoma cell specific markers to allow tumor cell identification. Figure 17 shows the expression levels per indication.

Patient-derived tumor cells were opsonized with bsIgG1-016-H5L2-LC90S-F405L×010-H5L2-K409R-E430G at 10 μg/mL or 100 μg/mL, and CDC induction was assessed in the presence of 20% pooled NHS. The following cell markers were used to identify the different cell populations: CD45-KO (Beckman Coulter B36294), CD19-PC7 (Beckman Coulter, cat. no. IM3628), CD3-V450 (BD, cat. no. 560365), CD5-APC (BD, cat. no. 345783), CD5-PE (DAKO, cat. no. R084201), CD10-APC-H7 (BD, cat. no. 655404), CD10-PE (DAKO, cat. no. R084201), CD23-FITC (Biolegend, cat. no. 338505), lambda-APC-H7 (BD, cat. no. 656648), kappa-PE (DAKO, cat. no. R043601) and lambda-FITC (Emelca Bioscience CYT-LAMBF). Within the CD45+ cell population, malignant B cells were determined by different markers depending on the indication: CD3-/CD19+/CD5+ (CLL), CD3-/CD19+/CD10+ (FL, DLBCL), CD3-/CD19+/CD5+/CD23- (MCL). If malignant B cells could not be identified based on these markers, malignant tumor cells were identified based on clonality using kappa/lambda staining. Also, in some samples, malignant B cells could not be identified based on clonality. In such cases, the total B cell population was evaluated without distinguishing between normal and malignant B cells. Killing was calculated as the fraction (%) of 7-aminoactinomycin D (7-AAD; BD, cat. no. 555816) positive malignant B cells measured by an LSRFortessa flow cytometer (BD Biosciences, San Jose, CA).

Figure 18 shows that bsIgG1-016-H5L2-LC90S-F405Lx010-H5L2-K409R-E430G was highly potent (>50% lysis) in inducing CDC in tumor cells from patients with CLL, FL, MCL, DLBCL or B-NHL (not further specified). In cells from one patient with relapsed/refractory FL, bsIgG1-016-H5L2-LC90S-F405Lx010-H5L2-K409R-E430G had a low ability to induce CDC.

Example 13: Binding of bispecific CD37 antibodies with Fc-Fc interaction enhancing mutations to human or cynomolgus monkey B cells in whole blood and induction of cytotoxicity in B cells in whole blood
Binding to human or cynomolgus monkey B cells
Binding to human or cynomolgus B cells was measured in a whole blood binding assay. Heparinized human blood from healthy volunteers was obtained from UMC Utrecht (Utrecht, The Netherlands) and hirudin-treated blood from cynomolgus monkeys was obtained from Covance (Munster, Germany). Blood was aliquoted (35 μL/well) into wells of a 96-well round-bottom plate (Greiner Bio-one, cat. no. 65010). Red blood cells (RBCs) were lysed by the addition of 100 μL of RBC lysis buffer (10 mM KHC0 ₃ [Sigma P9144], 0.1 mM EDTA [Fluka 03620] and 0.15 mM NH ₄ CL [Sigma A5666]) and incubated on ice until RBC lysis was complete. After centrifugation at 300×G for 3 min, cells were incubated for 30 min at 4°C with Alexa-488-labeled bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G or Alexa-488-labeled control IgG1 (IgG1-b12) and serial dilutions of directly labeled antibodies (0.014-30 μg/mL final antibody concentration in 3× serial dilutions) to identify B cells (in a mixture of antibodies to further identify blood cell subsets).

For human blood B cells, the following antibodies were used:

For cynomolgus blood B cells, the following antibodies were used:

Cells were pelleted, washed twice in 150 μL FACS buffer, and resuspended in 150 μL TO-PRO-3 (final concentration 0.2 μM; Molecular Probes, cat no. T3605). Samples were measured by flow cytometry using an LSRFortessa flow cytometer. Binding is expressed as the geometric mean of A488 fluorescence intensity for live TO-PRO-3 ⁻ /CD14 ⁻ /CD19 ⁺ B cells (human) or live TO-PRO-3 ⁻ /CD14 ⁻ /CD19 ⁺ /CD20 ⁺ B cells (cynomolgus monkey). Log-transformed data were analyzed using best fit values of a nonlinear dose-response fit in GraphPad PRISM.

Figure 19 shows the concentration-dependent binding of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G to blood B cells from (A) human and (B) cynomolgus monkeys as one representative donor/animal. The mean _EC50 values for binding to human and cynomolgus B cells were in the same range (0.85μg/mL±0.284 based on binding to blood B cells from 6 human donors and 0.63μg/mL±0.228 based on binding to blood B cells from 4 cynomolgus monkeys, respectively), indicating that bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G shows comparable binding to human and cynomolgus CD37.

Cytotoxicity against human or cynomolgus B cells Cytotoxicity against human or cynomolgus B cells was measured in a whole blood cytotoxicity assay. Hirudin-treated human blood from healthy volunteers was obtained from UMC Utrecht (Utrecht, The Netherlands) and hirudin-treated blood from cynomolgus monkeys was obtained from Covance (Munster, Germany). Blood was aliquoted into wells of a 96-well round-bottom plate at 35 μL/well.

Serial dilutions of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G or IgG1-b12 (0.0005-10 μg/mL final antibody concentration in 3x serial dilutions; final volume 100 μL/well) were added. In cytotoxicity assays using human whole blood, the monoclonal FcγR interaction-enhanced CD37-specific antibody IgG1-G28.1-S239D-I332E was included as a reference. Samples were incubated for 4 hours at 37°C. Red blood cells were then lysed as above and samples were stained to identify B cells as above. Cells were pelleted, washed twice in 150 μL FACS buffer and resuspended in 150 μL TO-PRO-3 (final concentration 0.2 μM; Molecular Probes, cat no. T3605). Samples were counted by flow cytometry using an LSRFortessa flow cytometer. After exclusion of doublets, the percentage of viable TO-PRO-3 ^- /CD14 ^- /CD19 ⁺ B cells (human) or viable TO-PRO-3 ^- /CD14 ^- /CD19 ⁺ /CD20 ⁺ B cells (cynomolgus monkey) was measured. Percentage of B cell depletion was calculated as follows: Percentage of B cell depletion = 100 x [(% B cells in no Ab control - % B cells in sample) / (% B cells in no Ab control)]. Log-transformed data were analyzed using the best fit value of a nonlinear dose-response fit in GraphPad PRISM.

Figure 20 shows the concentration-dependent cytotoxicity of bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G against blood B cells from (A) humans and (B) cynomolgus monkeys as one representative donor/animal.

Based on _EC50 , the ability of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G to induce cytotoxicity was comparable between human and cynomolgus B cells. The mean _EC50 of cytotoxicity against human B cells (in blood from six donors) was 0.077 μg/mL ± 0.039; the mean _EC50 of cytotoxicity against cynomolgus B cells (in blood from four animals) was 0.043 μg/mL ± 0.019.

Figure 20A also shows the cytotoxic effect of the monoclonal CD37 antibody IgG1-G28.1-S239D-I332E with enhanced FcγR interactions against human B cells in a representative responding donor, which showed a lower cytotoxic effect than bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G. A maximum B cell depletion of 50% was measured with IgG1-G28.1-S239D-I332E in B cells from three responding donors, whereas no cytotoxic effect was measured against B cells by this antibody in B cells from the other three donors. bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G induced cytotoxicity in 93-99% of B cells in 6/6 donors. Binding of IgG1-G28.1-S239D-I332E to CD37 expressed on Daudi cells was comparable to that of bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G (data not shown).

Example 14: Potent CDC activity by combination of a bispecific CD37 antibody with an Fc-Fc interaction enhancing mutation and a CD20-specific antibody
The combination of bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G with an anti-CD20 antibody (IgG1-CD20-ofa; ofatumumab) was tested for its ability to induce CDC in patient-derived CLL tumor cells obtained from ConversantBio (Huntsville, Alabama, USA). PBMCs from patients were resuspended in RPMI containing 0.2% BSA (bovine serum albumin) and plated at a density of ^0.1x106 cells/well (30μL/well) in polystyrene 96-well round bottom plates (Greiner bio-one Cat # 650101) and 50μL of a concentration series of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G (0.0625-0.05μg/mL) and IgG1-CD20-ofa (1-8μg/mL) were added in 2-fold dilutions. bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G and IgG1-CD20-ofa were combined by mixing two concentrations that would reach the same effect on average separately, with the antibody concentration based on the relative potency (EC50 difference) of each antibody. IgG1-b12 was used as a negative control.

After incubation (15 min at room temperature with shaking), 20 μL of pooled normal human serum (NHS Cat # M0008 Sanquin, Amsterdam, The Netherlands) was added to each well as a complement source and the plate was incubated at 37°C for 45 min. The reaction was stopped by chilling the plate on ice. After centrifugation at 300 × g for 3 min, the cells were washed twice with 150 μL of FACS buffer and incubated with R-phycoerythrin (PE)-conjugated mouse anti-human IgG1-CD19 antibody (clone J3-119, Beckman Coulter, cat no. A07769, diluted 1:50 from stock solution) for 30 min at 4°C to measure tumor B cells and TO-PRO-3 (final concentration 0.2 μM; Molecular Probes, cat no. T3605) for identification of dead cells. Cells were pelleted, washed twice in 150 μL of FACS buffer, and counted by flow cytometry using an LSRFortessa flow cytometer. The percentage of viable cells was calculated as follows: % viable cells = 100 x (number of TO-PRO-3 negative events)/(total number of events).

Figures 21A-D show that both bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G and ofatumumab induced CDC in tumor cells from two CLL patients, with enhanced CDC activity with increasing dose levels. Combining bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G with ofatumumab resulted in enhanced CDC activity in both CLL patients tested at all concentrations tested, although these effects were less pronounced at higher antibody concentrations where single agents induced nearly complete cell death (Figures 21A and B). These results indicate that the addition of ofatumumab to bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G can improve CDC-mediated tumor cell death in malignant B cells obtained from CLL patients.

Example 15: Antitumor activity of bispecific CD37 antibodies with Fc-Fc interaction enhancing mutations in xenograft models of B cell malignancies
Antitumor activity in a subcutaneous JVM-3 human chronic B-cell leukemia xenograft model
JVM-3 cells ( ^1x107 ) were inoculated into the right flank of CB17.SCID mice and antibody treatment (three weekly intravenous injections at doses of 0.1, 0.3, 1, 3, or 10 mg/kg; IgG1-b12 was used as a negative control and given at 10 mg/kg) was initiated when ^tumors reached an average volume of approximately 158 mm3. Tumor volumes were measured twice weekly in two dimensions using calipers and expressed in ^mm3 using the formula: V = (L x W x W)/2, where V is tumor volume, L is tumor length (longest tumor dimension), and W is tumor width (longest tumor dimension perpendicular to L).

Figure 22A shows tumor volume over time per treatment group, and Figure 22B shows tumor volume per mouse per treatment group at day 25, when all groups were still intact. Three weekly doses of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G at 1, 3, or 10 mg/kg significantly reduced JVM-3 cell tumor growth, whereas dosing at 0.1 or 0.3 mg/kg had no effect on tumor growth (Mann-Whitney test, p<0.01).

Antitumor activity in an intravenous Daudi-luc Burkitt's lymphoma xenograft model
On day 0, SCID mice (CB-17/IcrHan® Hsd-Prkdcscid; Harlan) were intravenously injected with Daudi-luc (luciferase-transfected Daudi cells, 2.5× ¹⁰⁶ cells/mouse). On days 14, 21 and 28, mice were intraperitoneally injected with bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G at 0.1, 0.3, 1, 3 or 10 mg/kg. IgG1-b12 was used as a negative control antibody and was administered at 10 mg/kg. Tumor growth was assessed weekly (starting on day 2) by bioluminescence imaging (BLI). Mice were injected intraperitoneally with 100 μL of firefly D-luciferin (30 mg/mL; Caliper LifeSciences, cat. no. 119222), and bioluminescence (radiance in p/s/ ^cm2 /sr [photons/second/ ^cm2 /square radian]) was measured using a Biospace Bioluminescence Imaging System (PerkinElmer; mice were photographed from the dorsal side) under isoflurane anesthesia.

Figure 23A shows luciferase activity (bioluminescence, as a measure of tumor volume) over time per treatment group. Figure 23B shows luciferase activity per mouse per treatment group at day 36, when all groups were still intact. Three weekly doses of bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G at 0.1, 0.3, 1, 3, or 10 mg/kg significantly reduced the in vivo growth of Daudi-luc cells (one-way ANOVA, Fisher's LSD uncorrected).

Example 16: Evaluation of plasma clearance of bispecific CD37 antibodies with Fc-Fc interaction enhancing mutations in SCID mice
Female SCID mice (CB-17/IcrHan® Hsd-Prkdcscid; Harlan) aged 11-12 weeks (3 mice per group) were injected intravenously (iv) once with 100 μg (5 mg/kg) or 500 μg (25 mg/kg) of bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G or IgG1-b12. As neither bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G nor IgG1-b12 showed cross-reactivity with mice, this experiment was designed to test antibody clearance in the absence of target-mediated clearance.

50-100 μL blood samples were collected from the saphenous vein at 10 min, 4 h, 24 h, 2 days, 7 or 8 days, 14 days, and 21 days after antibody administration. Blood was collected into heparin-containing vials and centrifuged at 10,000 g for 5 min. Plasma samples were diluted 1:50 (20 μL sample in 980 μL PBSA (PBS supplemented with 0.2% bovine serum albumin (BSA))) for mice administered 5 mg/kg and 1:20 (20 μL sample in 380 μL PBSA) for mice administered 25 mg/kg and stored at −20°C until measurement of mAb concentration.

Human IgG concentrations were measured using a sandwich ELISA. Mouse mAb anti-human IgG kappa clone MH16 (CLB Sanquin, The Netherlands; cat. no. M 1268) was used as capture antibody at a concentration of 2 μg/mL, with 100 μL coated overnight at 4°C on 96-well Microlon ELISA plates (Greiner, Germany). After blocking the plates with PBSA for 1 h at room temperature (RT), samples were added, serially diluted in PBSA, and incubated for 1 h at RT on a plate shaker. Plates were washed three times with 300 μL PBST (PBS supplemented with 0.05% Tween 20) and then incubated with goat anti-human IgG immunoglobulins (Jackson, West Grace, PA; cat. no. 109-035-098; 1:10.000 in PBST supplemented with 0.2% BSA) for 1 h at RT. Plates were washed again three times with 300 μL PBST and then incubated with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS; Roche, Mannheim, Germany) under light protection. The reaction was stopped by adding 100 μL 2% oxalic acid. Absorbance was measured at 405 nm in a microplate reader (Biotek, Winooski, VT). Human IgG concentrations were calculated by using the injected material as a reference curve. Purified human IgG1 (The binding site, cat. no. BP078) was included as a plate control. Human IgG concentrations (in μg/mL) were plotted (Figure 24A and C) and AUC (area under the curve) was calculated using Graphpad prism 6.0. IgG clearance was measured by the last day of blood sampling (day 21) by the formula D*1.000/AUC, where D is the injection dose (1 mg/kg) (Figures 24B and D).

There was no substantial difference in plasma clearance rates between bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G and IgG1-b12, demonstrating that bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G exhibits a pharmacokinetic profile comparable to wild-type human IgG1 in the absence of target binding.

Example 17: Determining the contribution of CD37 amino acid residues to binding of CD37 antibodies using alanine scanning
Library Design
A CD37 single residue alanine library was synthesized (Geneart) in which all amino acid (aa) residues in the extracellular domain of human CD37 (Uniprot P11049) were individually mutated to alanine, except for positions that already contained an alanine or cysteine. Cysteines were not mutated to minimize potential disruption of the antigen's structure. The library was cloned into a pMAC expression vector containing a CMV/TK-polyA expression cassette, an Amp resistance gene, and a pBR322 origin of replication.

Library generation and screening Wild-type CD37 and alanine mutants were individually expressed in Freestyle HEK293 cells according to the manufacturer's instructions (Thermo Scientific). One day after transfection, cells were harvested. Approximately 100,000 cells were incubated with 20 μL of Alexa488-conjugated bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G (monovalent conjugate 010) or Alexa488-conjugated bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G (monovalent conjugate 016) at a concentration of 3 μg/mL in FACS buffer (PBS + 0.1% (w/v) bovine serum albumin (BSA) + 0.02% (w/v) sodium azide). The cells were incubated for 1 hour at room temperature. Afterwards, the cells were washed twice by adding 150 μL of FACS buffer and removing the supernatant after centrifugation. The cells were resuspended in 20 μL of fresh FACS buffer and stored at 4° C. until analysis by flow cytometry using an iQue screener (IntelliCyt). The entire experiment was performed in duplicate.

式中、「aa位置」とは、CD37または野生型（wt）CD37中の特定のアラニン変異位置を指す。 Data Analysis : For each sample, the average antibody binding per cell was measured as the geometric mean of fluorescence intensity (gMFI) of the ungated cell population. gMFI is affected by the affinity of the antibody for the CD37 variant and the expression level of the CD37 variant per cell. Because specific alanine mutations can affect the surface expression level of variant CD37, and also to correct for expression differences between CD37 variants in general, data were normalized to the binding intensity of a non-competing CD37-specific control antibody (in this example, monovalent binding antibody 010 and monovalent binding antibody 016 were non-competing antibodies, and one antibody was used as a control for the other antibody). The following formula was used:

wherein "aa position" refers to a particular alanine mutation position in CD37 or wild-type (wt) CD37.

式中、μおよびσは、すべての変異体の正規化gMFIの平均値および標準偏差（SD）である。 To represent the loss or gain of antibody binding, a standard score was determined according to the following calculation:

where μ and σ are the mean and standard deviation (SD) of the normalized gMFI of all variants.

Gains in binding in many cases result from loss of binding of the reference antibody to the particular ala variant. Using these calculations, amino acid positions where there is no loss or gain of binding by a particular antibody when the amino acid is replaced with alanine will receive a z-score of "0", gains in binding will receive a "z-score >0", and losses in binding will receive a "z-score <0". To correct for sample-to-sample variability, only CD37 amino acid residues with z-scores below -1.5 were considered as "loss-of-binding variants". Data were excluded from the analysis if the gMFI of the control antibody for a particular CD37 variant was below the mean gMFI _{control Ab} mean gMFI - 2.5 x SD (such CD37 variants were considered to have insufficient expression levels).

Figure 25 shows the "z-score (fold change)" of CD37 antibodies against CD37 variants with ala mutations at positions 42-131 (according to SEQ ID NO: 94). The results show:
the binding of antibody 010 is dependent on at least aa Y182, D189, T191, I192, D194, K195, V196, I197, and P199 of human CD37;
- Binding of antibody 016 is dependent on at least aa E124, F162, Q163, V164, L165, and H175 of human CD37.

In summary, bispecific antibodies composed of two non-competing CD37-specific antibodies with Fc-Fc interaction enhancing mutations for binding to the target showed the most favorable combination of CDC and ADCC potency in CD37-positive tumor cells. With regard to both effector mechanisms, bispecific antibodies with Fc-Fc interaction enhancing mutations showed superior potency compared to the combination of two non-competing CD37 antibodies containing Fc-Fc interaction enhancing mutations or to a single CD37 antibody with an Fc-Fc interaction enhancing mutation.

Example 18: In vitro evaluation of the CDC activity of a mixture of a novel hexamerization-enhanced CD37 antibody and a clinically established CD20 antibody product on Raji cells
The CDC activity of a mixture of CD37 antibodies IgG1-37.3-E430G, IgG1-G28.1-E430G, IgG1-004-E430G, IgG1-005-E430G, IgG1-010-E430G and IgG1-016-E430G (the latter four are chimeric rabbit/human antibodies) with Fc-Fc interaction enhancing mutations plus the clinically established CD20 targeting monoclonal antibody products MabThera (rituximab; Roche, H0124B08), Arzerra (ofatumumab; Novartis; C656294) and Gazyva (obinutuzumab, GA101; Roche, D287-41A GACD20) was tested in vitro using Burkitt's lymphoma Raji cells. Raji cells (ATCC, Cat No. CCL-86) were cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS, 1 U/mL penicillin, 1 μg/mL streptomycin and 4 mM L-glutamine. 0.1× ¹⁰⁶ Raji cells were preincubated with antibodies in a total volume of 80 μL per well in RPMI/0.2% BSA for 15 min on a shaker at room temperature. NHS was then added to the preincubated cells to a final volume of 100 μL (final antibody concentration 10 μg/mL; 20% NHS) and incubated for 45 min at 37°C. For all total antibody concentrations tested, different ratios of the two antibodies in the mixture (1:0-3:1-1:1-1:3-0:1) were tested. The plates were centrifuged and the cells were resuspended in 30 μL PI (2 μg/mL). Killing was calculated as the fraction (%) of PI positive cells as measured by flow cytometry on an iQue screener (IntelliCyt). Data were analyzed and plotted using GraphPad Prism software.

The mixture of the tested CD37 antibodies with Fc-Fc interaction enhancing mutations and clinically established CD20 antibody products showed enhanced dose-dependent CDC activity against Raji cells compared to the same concentration of the antibodies alone (Figure 8). There was little difference in CDC activity at the different tested ratios of the two antibodies in the mixture (1:3, 1:1 or 3:1). These data indicate that a mixture of hexamerization-enhanced CD37 antibodies with Fc-Fc interaction enhancing mutations + clinically established CD20 antibody products, such as MabThera, Arzerra (type I CD20 antibodies) or Gazyva (type II CD20 antibodies), may improve therapeutic potential for patients with B-cell malignancies that are often refractory to standard CD20 targeting treatments alone.

Example 19: Antibody formulation studies Antibodies IgG1-010-H5L2-K409R-E430G (E1) and IgG1-016-H5L2-LC90S-F405L-E430G (D1) were each formulated in three different formulations with the following compositions:

(Table 4)

pH measurements were performed according to USP <791> pH. Of each of these formulations, 1.95 ml was transferred to a Nalgene cryo-tube and subjected to two freeze-thaw cycles consisting of freezing at -65°C for 12 hours and then thawing at 25°C for 12 hours. Samples were tested at time 0 and after two freeze/thaw cycles.

Visible Particles Visible particle counts were performed against a black background and against a white background with illumination of minimum luminance between 2000 and 3750 lux.

All three formulations of each of the two antibodies were nearly free of visible particles (0-3 particles/ml) both at time 0 and after freeze-thaw cycles. Thus, the samples were stable with respect to the formation of visible particles.

Turbidity Turbidity testing was performed by measurement against a pharmacopoeia reference standard solution using a turbidimeter. Sample solution results (in Nephelometric Turbidity Units (NTU)) were compared to those of the closest reference solution. Results were reported as equivalent to the reference solutions if the sample results were within [-10% to +10%] of the NTU value of the respective reference solution.

Turbidity values measured after two freeze-thaw cycles are shown in Figure 27. All turbidity values were low and within reference suspensions II and III. F1 showed the lowest turbidity, with the turbidity of antibody IgG1-016-H5L2-LC90S-F405L-E430G (D1) in F1 being slightly higher than that of antibody IgG1-010-H5L2-K409R-E430G (E1). Turbidity increased slightly with increasing NaCl.

Subvisible Particles
Subvisible particles after two freeze-thaw cycles were detected by the light obscuration principle using the HIAC instrument. Particles larger than 2, 5, 10, or 25 micrometers were counted.

Figure 28 shows that all three formulations for both antibodies contained only a small number of subvisible particles, especially very few particles larger than 10 or 25 micrometers. The number of subvisible particles was smallest in formulation F2.

Size Exclusion Chromatography (SEC)
Size-exclusion UPLC (SE-UPLC) was used to measure the amount of monomer, high molecular weight species (HMWS/aggregates), and low molecular weight species (LMWS/fragments) present in the samples. The method was performed on an Acquity UPLC Protein BEH SEC or equivalent column connected to a (U)HPLC system. Elution peaks were detected by absorbance at 280 nm. The main peak, HMWS, and LMWS are expressed as percentages (%) of relative peak areas.

The data are shown in the table below (LOQ indicates below the limit of quantification).

(Table 5)

(Table 6)

The data showed that total HMWS and total LMWS were low for both antibodies, and no significant increase in HMWS and LMWS was found after two cycles of freeze-thaw. There were no significant differences between the three formulations.

Dynamic Light Scattering (DLS)
The evaluation of the diffusion interaction parameter kD (ml/g) was performed via dynamic light scattering (DLS) using a DynaPro Plate Reader II (with software Dynamics; Wyatt) in 384-well plates. Serial dilutions of the proteins in various buffers were prepared. _Dm ( ^m2 /s; DLS-derived mutual diffusion coefficient) was plotted against the protein concentration c (g/mL). kD is obtained by dividing the slope calculated from the linear fit by the intercept, which is _D0 ( ^m2 /s; diffusion coefficient at infinite solute concentration).
_Dm = _D0 (1 + kD*c)
The kD values of the samples (not subjected to two freeze-thaw cycles) are shown in the table below.

(Table 7)

The data show only slightly attractive behavior of the antibody in the three formulations.

Overall, the three formulations of both antibodies showed suitable characteristics for pharmaceutical use.

Example 20: Evaluation of pH, excipients and surfactants under stress conditions pH and excipient screening of six formulations (F4-F9) with different pH and ionic strength were evaluated.

Chemicals and excipients

Table 8

Table 9

The table below lists the formulations evaluated.

・タンパク質は二重特異性CD37抗体（bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G）である。 (Table 10)

- The protein is a bispecific CD37 antibody (bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G).

Methods Bispecific CD37 antibodies as specified in Table 20.1 were subjected to buffer exchange and upconcentration to generate the formulations listed in Table 20.3. The formulations were produced by 1) buffer exchange to achieve the target buffer concentration and pH, followed by 2) concentration above the target concentration. By using standard dilution procedures, protein concentration, pH value, and density were measured during protein processing and utilized in the calculation of each formulation required. Protein concentration and pH of the final formulated solutions were measured and confirmed. All formulation solutions were filtered using 0.22 μm polyvinylidene fluoride (PVDF) membrane filters.

Primary packaging material was prepared as appropriate and each formulation was manually filled into 6R/20 mm glass Type I vials using aseptic technique with a target fill volume of 2.4 mL, stoppered with 20 mm bromobutyl rubber stoppers (injection stoppers) and sealed with 20 mm aluminum flip-off seals. Samples of all formulations were labeled and stored at each condition for stability testing.

Post-filtered formulations The pH, protein concentration, and osmolality of the formulated solutions for the post-filtered liquid formulations (F4-F9) were measured. Protein concentrations measured by UV spectrophotometer (A280) at early time points during short-term stability studies were also included.

The prepared solution was free of visible particles after filtration.

Short-term stability test
Analysis after 2 weeks of storage:
Good stability for all formulations was observed at long-term conditions (2-8°C), with denaturation observed after up to 2 weeks of storage at accelerated conditions (25°C) and more significant at stressed conditions (40°C). The most significantly affected quality attributes were charge heterogeneity and monomer content by HP-SEC. Minor clipping was also observed in the Caliper CE-SDS results after 2 weeks of storage at stressed conditions.

All formulations showed an increase in aggregate content under stress conditions. Exceptionally high levels of aggregates were observed for F4. A slight increase in fragmentation was also observed for all formulations after stress storage.

Changes in charge variants were observed under stress conditions. Low pH seemed to slow down the rate of basic variant increase, but a 10% increase was still observed in formulation F4 (pH 5.0) compared to the TO sample. The highest basic variant content was observed in F7 (pH 6.0).

Analysis after 4 weeks of storage:
The trend observed in the samples stored for 2 weeks could be confirmed. For charge variants, specifically, basic variants showed a clear pH dependency, with F4 at pH 5.0 showing the least basic variant formation. However, other quality attributes such as aggregation and clipping were not favorable for the F4 formulation. Formulations with pH 6.0 or higher showed high basic variant formation.

Formulations with pH 5.5 showed acceptable quality characteristics, among which formulation F5 showed the lowest basic variant formation.

BsIgG1-016-H5L2-LC90S-F405L-E430G x 010-H5L2-K409R-E430G has sequence liability for succinimide. Efforts were made in this study to develop a formulation that would protect the bispecific CD37 antibody from degradation under long-term storage conditions. Results from eight studies could show a pH dependency for succinimide formation (reflected as an increase in basic charge variants in the stability samples), with the pH 5.0 formulation (F4) showing the smallest increase in basic variant content with storage time. However, this formulation was considered unsuitable due to poor stability behavior with respect to properties such as aggregation and fragmentation. Furthermore, the results show that formulations with higher pH, e.g. pH 5.5, provide better stability in all the quality attributes tested.

The results from the stability study of Example 20 are shown below in Tables 11-18.

Table 11

Table 12

iCE：イメージキャピラリー電気泳動 Table 13

iCE: Image Capillary Electrophoresis

iCE：イメージキャピラリー電気泳動 (Table 14)

iCE: Image Capillary Electrophoresis

Table 15

Table 16

Table 17

Table 18

Claims (26)

a) bispecific antibodies;
b) histidine buffer;
c) 50-300 mM sugar and/or 50-300 mM polyol, and
d) 0.01 to 0.1% polysorbate 80
A pharmaceutical composition comprising:
the pH of the composition is between 4.5 and 6.8; and the bispecific antibody comprises a heavy chain set forth in SEQ ID NOs: 124 and 125 and a light chain set forth in SEQ ID NOs: 119 and 126, wherein the heavy chain set forth in SEQ ID NO: 124 forms an antigen-binding region with the light chain set forth in SEQ ID NO: 119, and the heavy chain set forth in SEQ ID NO: 125 forms an antigen-binding region with the light chain set forth in SEQ ID NO: 126.
The pharmaceutical composition. The pharmaceutical composition of claim 1, comprising 5-100 mg/mL of the bispecific antibody, e.g., 10-50 mg/mL, e.g., 10-30 mg/mL, e.g., 20 mg/mL of the bispecific antibody. The pharmaceutical composition according to any one of the preceding claims, comprising 10 to 100 mM histidine, such as 10 to 50 mM, such as 10 to 30 mM, such as 20 mM histidine. The pharmaceutical composition according to any one of the preceding claims, comprising a sugar that is sucrose, and preferably comprising 75-275 mM sucrose, such as 100-250 mM, e.g. 100 mM sucrose or 250 mM sucrose. 5. The pharmaceutical composition of claim 4 , which is polyol-free. 5. The pharmaceutical composition according to any one of claims 1 to 4, comprising a polyol which is sorbitol or mannitol, preferably comprising 75 to 275 mM sorbitol or 75 to 275 mM mannitol, such as 100 to 250 mM sorbitol or 100 to 250 mM mannitol, such as 100 mM sorbitol or 100 mM mannitol or 250 mM sorbitol or 100 mM mannitol. The pharmaceutical composition according to any one of the preceding claims, comprising 0.01-0.05% polysorbate 80, e.g. 0.01%-0.04%, e.g. 0.02% or 0.04% polysorbate 80. The pharmaceutical composition according to any one of the preceding claims, wherein the pH is 5.5 to 6.5, for example 5.5 or 6.5. The pharmaceutical composition of any one of the preceding claims further comprising sodium chloride, e.g., 25 to 250 mM sodium chloride, e.g., 100 to 150 mM sodium chloride, e.g., 100 mM or 150 mM sodium chloride. The pharmaceutical composition of any one of the preceding claims, further comprising arginine, e.g., 25 to 200 mM arginine, e.g., 50 to 100 mM arginine, e.g., 75 mM arginine. a) 20 mg/mL bispecific antibody, 20 mM histidine, 250 mM sucrose, and 0.02% or 0.04% polysorbate 80 at pH 5.5-6.5; or
b) 20 mg/mL of bispecific antibody, 20 mM histidine, 100 mM sucrose, 0.02% or 0.04% polysorbate 80, and 100-150 mM, preferably 100 mM, sodium chloride at pH 5.5-6.5; or
c) 20 mg/mL of bispecific antibody, 20 mM histidine, 100 mM sucrose, 0.02% or 0.04% polysorbate 80, 75 mM arginine, and 100-150 mM, preferably 100 mM, sodium chloride at pH 5.5-6.5; or
d) A pharmaceutical composition according to any one of the preceding claims comprising 20 mg/mL of the bispecific antibody, 20 mM histidine, 100 mM sucrose, 0.02% or 0.04% polysorbate 80, and 75 mM arginine at pH 5.5-6.5. In aqueous solution:
a) 20 mg/mL bispecific antibody, 20 mM histidine, 250 mM sucrose, and 0.02% or 0.04% polysorbate 80 at pH 5.5-6.5; or
b) 20 mg/mL of bispecific antibody, 20 mM histidine, 100 mM sucrose, 0.02% or 0.04% polysorbate 80, and 100-150 mM, preferably 100 mM, sodium chloride at pH 5.5-6.5; or
c) 20 mg/mL of bispecific antibody, 20 mM histidine, 100 mM sucrose, 0.02% or 0.04% polysorbate 80, 75 mM arginine, and 100-150 mM, preferably 100 mM, sodium chloride at pH 5.5-6.5; or
d) The pharmaceutical composition according to any one of the preceding claims, consisting of 20 mg/mL of the bispecific antibody, 20 mM histidine, 100 mM sucrose, 0.02% or 0.04% polysorbate 80, and 75 mM arginine at pH 5.5-6.5. The pharmaceutical composition of any one of the preceding claims, which has enhanced CDC, or enhanced CDC and ADCC effector function, compared to the same bispecific molecule not having the Fc-Fc interaction enhancing mutation. A pharmaceutical composition according to any one of the preceding claims for use as a medicine. A pharmaceutical composition according to any one of the preceding claims for use in the treatment of cancer or an autoimmune disease or an inflammatory disorder. A pharmaceutical composition according to any one of the preceding claims for use in the treatment of allergies, graft rejection, or B-cell malignancies, such as non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), mantle cell lymphoma (MCL), plasma cell leukemia (PCL), diffuse large B-cell lymphoma (DLBCL), or acute lymphoblastic leukemia (ALL). 17. The pharmaceutical composition for use according to any one of claims 14 to 16 , which is administered parenterally, for example subcutaneously, intramuscularly or intravenously. 18. A pharmaceutical composition for use according to any one of claims 14 to 17 in combination with one or more further therapeutic agents. The pharmaceutical composition for use according to any one of claims 14 to 18, wherein the one or more further therapeutic agents are selected from the group comprising doxorubicin, cisplatin, bleomycin, carmustine, cyclophosphamide, chlorambucil, bendamustine, vincristine, fludarabine, ibrutinib, and anti - CD20 antibodies, such as rituximab or ofatumumab. The additional therapeutic agent is
i) the VH CDR1 sequence set forth in SEQ ID NO: 75;
VH CDR2 sequence as set forth in SEQ ID NO: 76;
VH CDR3 sequence as set forth in SEQ ID NO: 77;
The VL CDR1 sequence set forth in SEQ ID NO: 79;
a VL CDR2 sequence which is a DAS, and
VL CDR3 sequence as set forth in SEQ ID NO: 80;
ii) the VH CDR1 sequence set forth in SEQ ID NO: 82;
VH CDR2 sequence as set forth in SEQ ID NO: 83;
VH CDR3 sequence as set forth in SEQ ID NO: 84;
VL CDR1 sequence as set forth in SEQ ID NO: 85;
a VL CDR2 sequence which is a DAS, and
VL CDR3 sequence as set forth in SEQ ID NO: 86;
iii) the VH CDR1 sequence set forth in SEQ ID NO: 95;
VH CDR2 sequence as set forth in SEQ ID NO: 96;
VH CDR3 sequence as set forth in SEQ ID NO: 97;
VL CDR1 sequence as set forth in SEQ ID NO: 99;
a VL CDR2 sequence which is an ATS, and
VL CDR3 sequence as set forth in SEQ ID NO: 100;
iv) the VH CDR1 sequence set forth in SEQ ID NO: 88;
VH CDR2 sequence as set forth in SEQ ID NO: 89;
VH CDR3 sequence as set forth in SEQ ID NO: 90;
VL CDR1 sequence as set forth in SEQ ID NO: 92;
a VL CDR2 sequence which is a DAS, and
The VL CDR3 sequence set forth in SEQ ID NO: 93; and
v) the VH CDR1 sequence set forth in SEQ ID NO: 102;
VH CDR2 sequence as set forth in SEQ ID NO: 103;
VH CDR3 sequence as set forth in SEQ ID NO: 104;
The VL CDR1 sequence set forth in SEQ ID NO: 106;
a VL CDR2 sequence that is QMS, and
20. The pharmaceutical composition for use according to claim 18 or claim 19 , which is an anti-CD20 antibody capable of binding to human CD20, comprising a CDR sequence selected from the group consisting of the VL CDR3 sequences set forth in SEQ ID NO: 107. Use of a pharmaceutical composition according to any one of claims 1 to 13 for the manufacture of a medicament. 22. The use according to claim 21 for the manufacture of a medicament for the treatment of cancer, an autoimmune disease, or an inflammatory disease. 22. Use according to claim 21 for the manufacture of a medicament for the treatment of allergies, transplant rejection, or B-cell malignancies, such as non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), mantle cell lymphoma (MCL), plasma cell leukemia (PCL), diffuse large B-cell lymphoma ( DLBCL ), or acute lymphoblastic leukemia (ALL). 24. The use according to any one of claims 21 to 23 , wherein the pharmaceutical composition is administered parenterally, for example subcutaneously, intramuscularly or intravenously. 25. The use according to any one of claims 21 to 24 in combination with one or more further therapeutic agents. 26. The use of claim 25, wherein the one or more further therapeutic agents are selected from the group comprising doxorubicin, cisplatin, bleomycin, carmustine, cyclophosphamide, chlorambucil, bendamustine, vincristine, fludarabine, ibrutinib, and anti- CD20 antibodies, such as rituximab or ofatumumab.

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